UNIT 1 - Molecules and their Interaction Relevant to Biology

1.1 MOLECULES AND THEIR INTERACTION RELAVENT TO BIOLOGY

1.1.1 Structure of atoms, molecules and chemical bonds

Snapshot

Atoms are the smallest entities with which we often come across

The atoms contain, Protons, Neutrons, electrons and many more subatomic particles.

Molecules are the aggregates of the atoms in a particular fashion

The aggregation calls for a bonding: Chemical bonds

are many types of bonds, namely Covalent, Electrostatic, Coordinate, Ionic, Vander Waal’s forces,…

1.1.1.1 Structure of the Atom

The atom is a basic unit of matter that consists of a dense central nucleus surrounded by a cloud of negatively charged electrons. The atomic nucleus contains a mix of positively charged protons and electrically neutral neutrons (except for hydrogen-1, which is the only stable nuclide with no neutrons). The electrons of an atom are bound to the nucleus by the electromagnetic force. Likewise, a group of atoms can remain bound to each other by chemical bonds based on the same force, forming a molecule. An atom containing an equal number of protons and electrons is electrically neutral, otherwise it is positively or negatively charged and is known as an ion. An atom is classified according to the number of protons and neutrons in its nucleus: the number of protons determines the chemical element, and the number of neutrons determines the isotope of the element.

Chemical atoms, which in science now carry the simple name of "atom," are minuscule objects with diameters of a few tenths of a nanometre and tiny masses proportional to the volume implied by these dimensions. Atoms can only be observed individually using special instruments such as the scanning tunnelling microscope. Over 99.94% of an atom's mass is concentrated in the nucleus. Protons and neutrons having roughly equal mass. Each element has at least one isotope with an unstable nucleus that can undergo radioactive decay. This can result in a transmutation that changes the number of protons or neutrons in a nucleus. Electrons that are bound to atoms possess a set of stable energy levels, or orbitals, and can undergo transitions between them by absorbing or emitting photons that match the energy differences between the levels. The electrons determine the chemical properties of an element, and strongly influence an atom's magnetic properties. The principles of quantum mechanics have been successfully used to model the observed properties of the atom.

In the above table we have used a unit of mass called the atomic mass unit (amu). This unit is much more convenient to use than grams for describing masses of atoms. It is defined so that both protons and neutrons have a mass of approximately 1 amu. Its precise definition will be given later. The important points to keep in mind are as follows:

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Protons and neutrons have almost the same mass, while the electron is approximately 2000 times lighter.

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Protons and electrons carry charges of equal magnitude, but opposite charge. Neutrons carry no charge (they are neutral).

It was once thought that protons, neutrons and electrons were spread out in a rather uniform fashion to form the atom (see J.J. Thompson’s plum pudding model of the atom on page 42), but now we know the actual structure of the atom to be quite different.

What does an atom look like?

Protons and neutrons are held together rather closely in the centre of the atom. Together they make up the nucleus, which accountsfor nearly all of the mass of the atom. Electrons move rapidly around the nucleus and constitute almost the entire volume of the atom. Although quantum mechanics are necessary to explain the motion of an electron about the nucleus, we can say that the distribution of electrons about an atom is such that the atom has a spherical shape.

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Atoms have sizes on the order of 1-5 A (1 angstrom = 1 A = 1 x 10-10 m) and masses on the order of 1-300 amu.

To put the mass and dimensions of an atom into perspective consider the following analogies. If an atom were the size of Ohio stadium, the nucleus would only be the size of a small marble. However, the mass of that marble would be ~ 115 million tons. What holds an atom together?

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Figure 1.1 A generic atomic planetary

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The negatively charged electron is attracted to the positively charged nucleus by a Columbic attraction.

The protons and neutrons are held together in the nucleus by the strong nuclear force. How many electrons, protons and neutrons are contained in an atom?

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Atoms in their natural state have no charge that is they are neutral. Therefore, in a neutral atom the number of protons and electrons are the same. If this condition is violated the atom has a net charge and is called an ion.

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The number of protons in the nucleus determines the identity of the atom. For example all carbon atoms contain six protons, all gold atoms contain 79 protons, and all lead atoms contain 82 protons.

Two atoms with the same number of protons, but different numbers of neutrons are called isotopes. How does the structure of the atom relate to its properties?

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Chemical reactions involve either the transfer or the sharing of electrons between atoms. Therefore, the chemical reactivity/ properties of an element is primarily dependent upon the number of electrons in an atom of that element. \Protons also play a significant role because the tendency for an atom to either lose, gain or share electrons is dependent upon the charge of the nucleus.

 Therefore, we can say that the chemical reactivity of an atom is dependent upon the number of electrons and protons, and independent of the number of neutrons.

The mass and radioactive properties of an atom are dependent upon the number of protons and neutrons in the nucleus.

Note: The number of protons, neutrons and electrons in an atom completely determine its properties and identity, regardless of how and where the atom was made. So it is inaccurate to speak of synthetic atoms and natural atoms. In other words a lead atom is a lead atom, end of story. It doesn’t matter if was mined from the earth, produced in a nuclear reactor, or came to earth on an asteroid.

Symbolism

There is a symbolism or shorthand for describing atoms which is universally used across all scientific disciplines

Atomic Number (Z) The # of protons

Mass Number (A) [The # of protons] + [the # of neutrons]

The number of protons, neutrons and electrons in an atom are uniquely specified by the following symbol

SyC

Where: Sy = the elemental symbol (i.e. C, N, Cr) defines the # of protons

A = the mass number [# of protons] + [# of neutrons]

C = the net charge [# of protons] [# of electrons]

1.1.1.2 Radioactive decay

Every element has one or more isotopes that have unstable nuclei that are subject to radioactive decay, causing the nucleus to emit particles or electromagnetic radiation. Radioactivity can occur when the radius of a nucleus is large compared with the radius of the strong force, which only acts over distances on the order of 1 fm.

The most common forms of radioactive decay are

Alpha decay is caused when the nucleus emits an alpha particle, which is a helium nucleus consisting of two protons and two neutrons. The result of the emission is a new element with a lower atomic number.

Beta decay (and electron capture) are regulated by the weak force, and result from a transformation of a neutron into a proton, or a proton into a neutron. The first is accompanied by the emission of an electron and an antineutrino, while the second causes the emission of a positron and a neutrino. The electron or positron emissions are called beta particles. Beta decay either increases or decreases the atomic number of the nucleus by one. An analog of positron beta decay in nuclei that are proton-rich is electron capture, a process even more common than positron emission since it requires less energy. In this type of decay an electron is absorbed by the nucleus, rather than a positron emitted. A neutrino is still emitted in this process, and a proton again changes to a neutron.

 Gamma decay results from a change in the energy level of the nucleus to a lower state, resulting in the emission of electromagnetic radiation. This can occur following the emission of an alpha or a beta particle form radioactive decay.

Other more rare types of radioactive decay include ejection of neutrons or protons or clusters of nucleons from a nucleus, or more than one beta particle, or result (through internal conversion) in production of highspeed electrons that are not beta rays, and high-energy photons that are not gamma rays. A few large nuclei explode into two or more charged fragments of varying masses plus several neutrons, in a decay called spontaneous nuclear fission. Each radioactive isotope has a characteristic decay time period the half-life that is determined by the amount of time needed for half of a sample to decay. This is an exponential decay process that steadily decreases the proportion of the remaining isotope by 50% every half-life. Hence after two half-lives have passed only 25% of the isotope is present, and so forth

1.1.2 Molecules

A molecule is an electrically neutral group of two or more atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their lack of electrical charge. However, in quantum physics, organic chemistry, and biochemistry, the term molecule is often used less strictly, also being applied to polyatomic ions.

In the kinetic theory of gases, the term molecule is often used for any gaseous particle regardless of its composition. According to this definition, noble gas atoms are considered molecules despite being composed of a single non-bonded atom.

A molecule is an aggregate of atoms that possesses distinctive observable properties

A molecule may be homonuclear, that is, it consists of atoms of a single chemical element, as with oxygen (O2); or it may be a chemical compound composed of more than one element, as with water (H2O).

Atoms and complexes connected by non-covalent bonds such as hydrogen bonds or ionic bonds are generally not considered single molecules

Molecules as components of matter are common in organic substances (and therefore biochemistry). They also make up most of the oceans and atmosphere. However, the majority of familiar solid substances on Earth, including most of the minerals that make up the crust, mantle, and core of the Earth, contain many chemical bonds, but are not made of identifiable molecules.

Also, no typical molecule can be defined for ionic crystals (salts) and covalent crystals (network solids), although these are often composed of repeating unit cells that extend either in a plane (such as in graphene) or three-dimensionally (such as in diamond, quartz, or sodium chloride).

The theme of repeated unit-cellular-structure also holds for most condensed phases with metallic bonding, which means that solid metals are also not made of molecules. In glasses (solids that exist in a vitreous disordered state), atoms may also be held together by chemical bonds without presence of any definable molecule, but also without any of the regularity of repeating units that characterises crystals.

1.1.2.1 Structure of molecules

The three dimensional shape or configuration of a molecule is an important characteristic. This shape is dependent on the preferred spatial orientation of covalent bonds to atoms having two or more bonding partners. Three dimensional configurations are best viewed with the aid of models. In order to represent such configurations on a two-dimensional surface (paper, blackboard or screen), we often use perspective drawings in which the direction of a bond is specified by the line connecting the bonded atoms. In most cases the focus of configuration is a carbon atom so the lines specifying bond directions will originate there. As defined in the diagram on the right, a simple straight line represents a bond lying approximately in the surface plane. The two bonds to substituents A in the structure on the left are of this kind. A wedge shaped bond is directed in front of this plane (thick end toward the viewer), as shown by the bond to substituent B; and a hatched bond is directed in back of the plane (away from the viewer), as shown by the bond to substituent D. Some texts and other sources may use a dashed bond in the same manner as we have defined the hatched bond, but this can be confusing because the dashed bond is often used to represent a partial bond (i.e. a covalent bond that is partially formed or partially broken). The following examples make use of this notation, and also illustrate the importance of including non-bonding valence shell electron pairs when viewing such configurations.

Bonding configurations are readily predicted by valence-shell electron-pair repulsion theory, commonly referred to as VSEPR in most introductory chemistry texts. This simple model is based on the fact that electrons repel each other, and that it is reasonable to expect that the bonds and non-bonding valence electron pairs associated with a given atom will prefer to be as far apart as possible. The bonding configurations of carbon are easy to remember, since there are only three categories.

In the three examples shown in table 1.2, the central atom (carbon) does not have any non-bonding valence electrons; consequently the configuration may be estimated from the number of bonding partners alone. For molecules of water and ammonia, however, the non-bonding electrons must be included in the calculation. In each case there are four regions of electron density associated with the valence shell so that a tetrahedral bond angle is expected. The measured bond angles of these compounds (H2O 104.5º & NH3 107.3º) show that they are closer to being tetrahedral than trigonal or linear. Of course, it is the configuration of atoms (not electrons) that defines the shape of a molecule, and in this sense ammonia is said to be pyramidal (not tetrahedral). The compound boron trifluoride, BF3, does not have non-bonding valence electrons and the configuration of its atoms is trigonal.

The best way to study the three-dimensional shapes of molecules is by using molecular models. Many kinds of model kits are available to students and professional chemists. Some of the useful features of physical

models can be approximated by the model viewing applet Jmol. This powerful visualization tool allows the user to move a molecular structure in any way desired. Atom distances and angles are easily determined. To measure a distance, double-click on two atoms. To measure a bond angle, do a double-click, single-click, double-click on three atoms. To measure a torsion angle, do a double-click, single-click, single-click, doubleclick on four atoms. A pop-up menu of commands may be accessed by the right button on a PC or a controlclick on a Mac while the cursor is inside the display frame.

One way in which the shapes of molecules manifest themselves experimentally is through molecular dipole moments. A molecule which has one or more polar covalent bonds may have a dipole moment as a result of the accumulated bond dipoles. In the case of water, we know that the O-H covalent bond is polar, due to the different electronegativities of hydrogen and oxygen. Since there are two O-H bonds in water, their bond dipoles will interact and may result in a molecular dipole which can be measured. The following diagram shows four possible orientations of the O-H bonds.

In the linear configuration (bond angle 180º) the bond dipoles cancel, and the molecular dipole is zero. For other bond angles (120 to 90º) the molecular dipole would vary in size, being largest for the 90º configuration. In a similar manner the configurations of methane (CH4) and carbon dioxide (CO deduced from their zero molecular dipole moments. Since the bond dipoles have cancelled, the configurations of these molecules must be tetrahedral (or squareplanar) and linear respectively.

Table 1.2 Structure of different molecules according to

The case of methane provides insight to other arguments that have been used t confirm its tetrahedral configuration. For purposes of discussion we shall consider three other configurations for CH Substitution of one hydrogen by a chlorine atom gives a CH Cl compound. Since the tetrahedral, square-

planar and square-pyramidal configurations have structurally equivalent hydrogen atoms, they would each give a single substitution product. However, in the trigonal-pyramidal configuration one hydrogen (the apex) is structurally different from the other three (the pyramid base). Substitution in this case should give two different CH3Cl compounds if all the hydrogen react. In the case of di-substitution, the tetrahedral configuration of methane would lead to a single CH2Cl2 product, but the other configurations would give two different CH2Cl2 compounds. These substitution possibilities are shown in the above insert.

1.1.2 Chemical bonds

A chemical bond is an attraction between atoms. This attraction may be seen as the result of different behaviours of the outermost electrons of atoms. Although all of these behaviours merge into each other seamlessly in various bonding situations so that there is no clear line to be drawn between them, nevertheless behaviours of atoms become so qualitatively different as the character of the bond changes quantitatively, that it remains useful and customary to differentiate between the bonds that cause these different properties of condensed matter.

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'Covalent' bond, one or more electrons (often a pair of electrons) are drawn into the space between the two atomic nuclei.

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Here the negatively charged electrons are attracted to the positive charges of both nuclei, instead of just their own. This overcomes the repulsion between the two positively charged nuclei of the two atoms, and so this overwhelming attraction holds the two nuclei in a fixed configuration of equilibrium, even though they will still vibrate at equilibrium position. Thus, covalent bonding involves sharing of electrons in which the positively charged nuclei of two or more atoms simultaneously attract the negatively charged electrons that are being shared between them.

 These bonds exist between two particular identifiable atoms, and have a direction in space, allowing them to be shown as single connecting lines between atoms in drawings, or modelled as sticks between spheres in models. In a polar covalent bond, one or more electrons are unequally shared between two nuclei.

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Covalent bonds often result in the formation of small collections of better-connected atoms called molecules, which in solids and liquids are bound to other molecules by forces that are often much weaker than the covalent bonds that hold the molecules internally together. Such weak intermolecular bonds give organic molecular substances, such as waxes and oils, their soft bulk character, and their low melting points (in liquids, molecules must cease most structured or oriented contact with each other). When covalent bonds link long chains of atoms in large molecules, however (as in polymers such as nylon), or when covalent bonds extend in networks though solids that are not composed of discrete molecules (such as diamond or quartz or the silicate minerals in many types of rock) then the structures that result may be both strong and tough, at least in the direction oriented correctly with networks of covalent bonds. Also, the melting points of such covalent polymers and networks increase greatly.

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Ionic bond, the bonding electron is not shared at all, but transferred. In this type of bond, the outer atomic orbital of one atom has a vacancy which allows addition of one or more electrons. These newly added electrons potentially occupy a lower energy-state (effectively closer to more nuclear charge) than they experience in a different atom. Thus, one nucleus offers a more tightly bound position to an electron than does another nucleus, with the result that one atom may transfer an electron to the other. This transfer causes one atom to assume a net positive charge, and the other to assume a net negative charge. The bond then results from electrostatic attraction between atoms, and the atoms become positive or negatively charged ions. Ionic bonds may be seen as extreme examples of polarization in covalent bonds. Often, such bonds have no particular orientation in space, since they

Therearetwocommon(andequivalent)ways describemolecularmass;bothare used relativemolecularmass,denoted Mr of a molecule Mr is a ratio, molecularmass, denotedm.This themolarmassdivided by Avogadro’s number.Themolecularmass, m, is expressed in daltons(abbreviated Da).Onedalton is equivalent to one-twelfththemass of carbon-12;akilodalton(kDa) is 1,000 daltons;amegadalton(MDa) is 1milliondaltons.Consider,forexample,a moleculewithamass1,000 timesthat of water. We cansay of thismoleculeeither Mr =18,000 or m= 18,000daltons. We can alsodescribe it asan “18 kDamolecule.” However,theexpression Mr 18,000 daltons is incorrect.Anotherconvenientunitfor describingthemass of asingleatom or molecule is theatomicmassunit(formerly amu,nowcommonlydenoted u).Oneatomicmassunit(1 u) is defined as onetwelfththemass of an atom of carbon-12.Sincetheexperimentallymeasuredmass of an atom of carbon-12 is 1.9926 x 1023 g, 1u=1.6606 x 10 24 g.Theatomicmass unit is convenientfordescribingthemass of a peak observed by massspectrometry

result from equal electrostatic attraction of each ion to all ions around them. Ionic bonds are strong (and thus ionic substances require high temperatures to melt) but also brittle, since the forces between ions are short-range, and do not easily bridge cracks and fractures. This type of bond gives a charactistic physical character to crystals of classic mineral salts, such as table salt.

Metallic bond. In this type of bonding, each atom in a metal donates one or more electrons to a "sea" of electrons that reside between many metal atoms. In this sea, each electron is free (by virtue of its wave nature) to be associated with a great many atoms at once. The bond results because the metal atoms become somewhat positively charged due to loss of their electrons, while the electrons remain attracted to many atoms, without being part of any given atom. Metallic bonding may be seen as an extreme example of delocalization of electrons over a large system of covalent bonds, in which every atom participates. This type of bonding is often very strong (resulting in the tensile strength of metals). However, metallic bonds are more collective in nature than other types, and so they allow metal crystals to more easily deform, because they are composed of atoms attracted to each other, but not in any particularly-oriented ways. This results in the malleability of metals. The sea of electrons in metallic bonds causes the characteristically good electrical and thermal conductivity of metals, and also their "shiny" reflection of most frequencies of white light.

Aromatic (aryl) compounds

Table 1.3Typical bond lengths in pm and bond energies in kJ/mol. Bond lengths can be converted to Å by division by 100(1Å= 100 pm).

Bond Length (pm) Energy (kJ/mol)

Hydrogen

436

Carbon

Coplanar structure, with all the contributing atoms in the same plane

atoms arranged in one or more rings

A number of π delocalized electrons that is even, but not a multiple of 4. That is, 4n + 2 number of π electrons, where n=0, 1, 2, 3, and so on. This is known as Hückel's Rule.

All bonds can be explained by quantum theory, but, in practice, simplification rules allow chemists to predict the strength, directionality, and polarity of bonds. The octet rule and VSEPR theory are two examples. More sophisticated theories are valence bond theory which includes orbital hybridization and resonance, and the linear combination of atomic orbitals molecular orbital method which includes ligand field theory. Electrostatics are used to describe bond polarities and the effects they have on chemical substances.

Figure1.7 Benzene, the mostwidely recognized aromatic compoundwith six(4n+2,n= 1)delocalized electrons.

Critical thinking Questions

1. What is theapproximatepercentage (in mass) of water in thehumanbody? Is this percentageexpected to be larger in theadult or in theoldindividual?

2. Canthe heat capacity of water be consideredsmall or large?What is thebiological significance of thatcharacteristic

There are four major classes of biomolecules - carbohydrates, proteins, nucleotides, and lipids.

Carbohydrates, or saccharides, are the most abundant of the four. Carbohydrates have several roles in living organisms, including energy transportation, as well as being structural components of plants and arthropods. Carbohydrate derivatives are actively involved in fertilization, immune systems, and the development of disease, blood clotting and development.

Carbohydrates are called carbohydrates because the carbon, oxygen and hydrogen they contain are generally in proportion to form water with the general formula Cn (H2O)n.

Carbohydrates (saccharides) - Molecules consist of carbon, hydrogen and oxygen atoms. A major food source and a key form of energy for most organisms. When combined together to form polymers, carbohydrates can function as long term food storage molecules, as protective membranes for organisms and cells, and as the main structural support for plants and constituents of many cells and their contents.

Lipids (fats) - Molecules consist of carbon, hydrogen, and oxygen atoms. The main constituents of all membranes in all cells (cell walls), food storage molecules, intermediaries in signaling pathways, Vitamins A, D, E and K, cholesterol.

Proteins - Molecules contain nitrogen, carbon, hydrogen and oxygen. They act as biological catalysts (enzymes), form structural parts of organisms, participate in cell signal and recognition factors, and act as molecules of immunity. Proteins can also be a source of fuel.

Nucleic acids (nucleotides) - DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). These molecules are involved in genetic information, as well as forming structure within cells. They are involved in the storage of all heritable information of all organisms, as well as the conversion of this data into proteins.

1.2.1 Carbohydrates

Carbohydrates are one of the main types of nutrients. They are the most important source of energy for your body. Your digestive system changes carbohydrates into glucose (blood sugar). Your body uses this sugar for energy for your cells, tissues and organs. It stores any extra sugar in your liver and muscles for when it is needed.

Carbohydrates are called simple or complex, depending on their chemical structure. Simple carbohydrates include sugars found naturally in foods such as fruits, vegetables, milk, and milk products. They also include sugars added during food processing and refining. Complex carbohydrates include whole grain breads and cereals, starchy vegetables and legumes. Many of the complex carbohydrates are good sources of fibre. For a healthy diet, limit the amount of added sugar that you eat and choose whole grains over refined grains.

The term is most common in biochemistry, it is called saccharide. The carbohydrates (saccharides) are divided into four chemical groupings:

1. monosaccharide

2. disaccharide

3. oligosaccharide

4. polysaccharide

In general, the monosaccharides and disaccharides, which are smaller (lower molecular weight) carbohydrates, are commonly referred to as sugars. While the scientific nomenclature of carbohydrates is complex, the names of the monosaccharides and disaccharides very often end in the suffix-ose. For example, grape sugar is the monosaccharide glucose, cane sugar is the disaccharide sucrose, and milk sugar is the disaccharide lactose.

1.2.1.1 Structure and Function

Formerly the name "carbohydrate" was used in chemistry for any compound with the formula Cm (H2O) n. Following this definition, some chemists considered formaldehyde (CH O) to be the simplest carbohydrate while others claimed that title for glyceraldehyde Today the term is generally understood in the biochemistry sense, which excludes compounds with only one or two carbons.

Monosaccharides

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Natural saccharides are generally built of simple carbohydrates called monosaccharides with general formula (CH

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A typical monosaccharide has the structure H-(CHOH)

H, that is, an aldehyde or ketone with many hydroxyl groups added, usually one on each carbon atom that is not part of the aldehyde or ketone functional group.

 Examples of monosaccharides are glucose, fructose, and glyceraldehyde. However, some biological substances commonly called "monosaccharides" do not conform to this formula (e.g., uronic acids and deoxy-sugars such as fucose), and there are many chemicals that do conform to this formula but are not considered to be monosaccharides (e.g., formaldehyde CH

The open-chain form of a monosaccharide often coexists with a closed ring form where the aldehyde/ketone carbonyl group carbon (C=O) and hydroxyl group (-OH) react forming a hemiacetal with a new C-O-C bridge.

Haworth projection

The three-dimensional structure of a monosaccharides in cyclic form is usually represented by its Haworth projection.

In the diagram 1.7B, the anomeric carbon below the plane of the carbon atoms, and the β-isomer has the -OH of the anomeric carbon above the plane.

Pyranoses typically adopt a chair conformation, similar to cyclohexane. In this conformation, the α-isomer has the -OH of the anomeric carbon in an axial position, whereas the β-isomer has the OH- of the anomeric carbon in equatorial position.

Disaccharides

Figure 1.7 B Fructose, a common sweatner

When the alcohol component of a glycoside is provided by a hydroxyl function on another monosaccharide, the compound is called a disaccharide.

Ex: Cellobiose : 4-O-β-D-Glucopyranosyl-D-glucose (the beta-anomer is drawn)

Maltose : 4-O-α-D-Glucopyranosyl-D-glucose (the beta-anomer is drawn)

Gentiobiose : 6-O-β-D-Glucopyranosyl-D-glucose (the alpha-anomer is drawn)

Trehalose : α-D-Glucopyranosyl-α-D-glucopyranoside

Although all the disaccharides shown here are made up of two glucopyranose rings, their properties differ in interesting ways. Maltose, sometimes called malt sugar, comes from the hydrolysis of starch. It is about one third as sweet as cane sugar (sucrose), is easily digested by humans, and is fermented by yeast. Cellobiose is obtained by the hydrolysis of cellulose.

It has virtually no taste, is indigestible by humans, and is not fermented by yeast. Some bacteria have beta-glucosidase enzymes that hydrolyse the glycosidic bonds in Cellobiose and cellulose. The presence of such bacteria in the digestive tracts of cows and termites permits these animals to use cellulose as a

The individual glucopyranoseringsarelabeledAand lightblue.Noticethattheglycosidebondmaybealpha,as in maltoseandtrehalose, thesedisaccharidesyieldsglucose as the onlyproduct.Enzyme-catalyzedhydrolysis alpha-glycosidasecleavesmaltoseand trehalose to gentiobiose.Abeta-glycosidasehastheoppositeactivity. In order to drawarepresentativestructureforcellobiose, thisfeature is often omitted in favor cellobioseand maltose is fromtheanomericcarbon ringBfree, so cellobioseandmaltosebothmayassumealphaandbetaanomers .Gentiobiosehasa beta-glycosidelink,originating thediagram.Because cellobiose,maltoseandgentiobiosearehemiacetalstheyareallreducingsugars(oxidized Tollen'sreagent).Trehalose,adisaccharide foundincertainmushrooms, -reducingsugar.Asystematicnomenclaturefordisaccharidesexists, but asthefollowingexamplesillustrate,theseareoftenlengthy.

Figure1.8AFourexamples

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food. Finally, it may be noted that trehalose has a distinctly sweet taste, but gentiobiose is bitter.

Disaccharides made up of other sugars are known, but glucose is often one of the components. Two important examples of such mixed disaccharides will be displayed above by clicking on the diagram. Lactose, also known as milk sugar, is a galactose-glucose compound joined as a beta-glycoside.

It is a reducing sugar because of the hemiacetal function remaining in the glucose moiety. Many adults, particularly those from regions where milk is not a dietary staple, have a metabolic intolerance for lactose.

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Infants have a digestive enzyme which cleaves the beta-glycoside bond in lactose, but production of this enzyme stops with weaning. Cheese is less subject to the lactose intolerance problem, since most of the lactose is removed with the whey.

 Sucrose, or cane sugar, is our most commonly used sweetening agent. It is a non-reducing

Figure1.9Lactose is adisaccharide found in milk. It consists of amolecule of D-galactose andamolecule of D-glucosebonded by beta1-4glycosidiclinkage. It hasaformula of

disaccharide composed of glucose and fructose joined at the anomeric carbon of each by glycoside bonds (one alpha and one beta). In the formula shown here the fructose ring has been rotated 180º from its conventional perspective.

Polysaccharides (or oligosaccharides)

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Polysaccharides is an important class of biological polymers. Their function in living organisms is usually either structure- or storage-related.

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Starch (a polymer of glucose) is used as a storage polysaccharide in plants, being found in the form of both amylose and the branched amylopectin.

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In animals, the structurally similar glucose polymer is the more densely branched glycogen, sometimes called 'animal starch'. Glycogen's properties allow it to be metabolized more quickly, which suits the active lives of moving animals.

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Structural polysaccharides : Cellulose , pectin and chitin.

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Cellulose is used in the cell walls of plants and other organisms, and is said to be the most abundant organic molecule on earth. It has many uses such as a significant role in the paper and textile industries, and is used as a feedstock for the production of rayon (via the viscose process), cellulose acetate, celluloid, and nitrocellulose.

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Chitin has a similar structure, but has nitrogen-containing side branches, increasing its strength. It is found in arthropod exoskeletons and in the cell walls of some fungi. It also has multiple uses, including surgical threads.

Polysaccharides also include callose or laminarin, chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan and galactomannan.

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Monosaccharides can be linked together into polysaccharides (or oligosaccharides) in a large variety of ways.

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Many carbohydrates contain one or more modified monosaccharide units that have had one or more groups replaced or removed.

Pectins are a family of complex polysaccharides that contain 1, 4- -D-galactosyluronic acid residues. They are present in most primary cell walls and in the non-woody parts of terrestrial plants.

Storage polysaccharide - Glycogen

Glycogen serves as the secondary long-term energy storage in animal and fungal cells, with the primary energy stores being held in adipose tissue. Glycogen is made primarily by the liver and

Glycogen is the analogue of starch, a glucose polymer in plants, and is sometimes referred to as animal starch, having a similar structure to amylopectin but more extensively branched and

Glycogen is a polymer of -linked branches.

Glycogen is found in the form of granules in the cytosol/cytoplasm in many cell types, and plays an important role in the glucose cycle. Glycogen forms an energy reserve that can be quickly mobilized to meet a sudden need for glucose, but one that is less compact than the less

In the liver hepatocytes, glycogen can compose up to eight percent (100 120 g in an adult) of the fresh weight soon after a meal. Only the glycogen stored in the liver can be made accessible to other organs. In the muscles, glycogen is found in a low concentration of one to two percent of the muscle mass.

The amount of glycogen stored in the body especially within the muscles, liver, and red blood cells varies with physical activity, basal metabolic rate, and eating habits such as intermittent fasting. Small amounts of glycogen are found in the kidneys, and even smaller amounts in certain glial cells in the brain and white blood cells. The uterus also stores glycogen during pregnancy, to nourish the embryo.

Glycogen is composed of a branched chain of glucose residues. It is stored in liver and muscles.

1. It is an energy reserve for animals.

2. It is the chief form of carbohydrate stored in animal body.

3. It is insoluble in water. It turns red when mixed with iodine.

4. It also yields glucose on hydrolysis.

Storage polysaccharide -Starch

Starches are glucose polymers in which glucopyranose units are bonded by alpha-linkages.

It is made up of a mixture of amylose (15 20%) and amylopectin (80 85%). Amylose consists of a linear chain of several hundred glucose molecules and Amylopectin is branched molecule made of several thousand glucose units (every chain of 24 glucose units is one unit of Amylopectin).

Starches are insoluble in water.

They can be digested by hydrolysis, catalysed by enzymes called amylases, which can break the alphalinkages (glycosidic bonds).

Humans and other animals have amylases, so they can digest starches. Potato, rice, wheat, and maize are major sources of starch in the human diet.

It is exclusively formed in plants and not in animals

1.2.2 Lipids

The lipids are a large and diverse group of naturally occurring organic compounds that are related by their solubility in nonpolar organic solvents (e.g. ether, chloroform, acetone & benzene) and general insolubility in water. Lipids include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, phospholipids, and others. The main biological functions of lipids include energy storage, signalling, and acting as structural components of cell membranes. Lipids have found applications in cosmetic and food industries as well as in nanotechnology

Fats, Oils, Waxes & Phospholipids

1. Fatty Acids

The common feature of these lipids is that they are all esters of moderate to long chain fatty acids. Acid or base-catalysed hydrolysis yields the component fatty acid, some examples of which are given in the following table, together with the alcohol component of the lipid. These long-chain carboxylic acids are generally referred to by their common names, which in most cases reflect their sources. Natural fatty acids may be saturated or unsaturated, and as the following data indicate, the saturated acids have higher melting points than unsaturated acids of corresponding size. The double bonds in the unsaturated compounds listed on the right are all cis (or Z).

The higher melting points of the saturated fatty acids reflect the uniform rod-like shape of their molecules. The cis-double bond(s) in the unsaturated fatty acids introduce a kink in their shape, which makes it more difficult to pack their molecules together in a stable repeating array or crystalline lattice. The trans-double

bond isomer of oleic acid, known as elaidic acid, has a linear shape and a melting point of 45 ºC (32 ºC higher than its cis isomer)

Two polyunsaturated fatty acids, linoleic and linolenic, are designated "essential" because their absence in the human diet has been associated with health problems, such as scaly skin, stunted growth and increased dehydration. These acids are also precursors to the prostaglandins, a family of physiologically potent lipids present in minute amounts in most body tissues.

Because of their enhanced acidity, carboxylic acids react with bases to form ionic salts, as shown in the following equations. In the case of alkali metal hydroxides and simple amines (or ammonia) the resulting salts have pronounced ionic character and are usually soluble in water. Heavy metals such as silver, mercury and lead form salts having more covalent character (3rd example), and the water solubility is reduced, especially for acids composed of four or more carbon atoms

2.Fats and Oils

The triesters of fatty acids with glycerol (1, 2, 3-trihydroxypropane) compose the class of lipids known as fats and oils. These triglycerides (or triacylglycerols) are found in both plants and animals, and compose one of the major food groups of our diet. Triglycerides that are solid or semisolid at room temperature are classified as fats, and occur predominantly in animals. Those triglycerides that are liquid are called oils and originate chiefly in plants, although triglycerides from fish are also largely oils

As might be expected from the properties of the fatty acids, fats have a predominance of saturated fatty acids, and oils are composed largely of unsaturated acids.

Thus, the melting points of triglycerides reflect their composition, as shown by the following examples. Natural mixed triglycerides have somewhat lower melting points, the melting point of lard being near 30 º C, whereas olive oil melts near -6 º C.

Since fats are valued over oils by some Northern European and North American populations, vegetable oils are extensively converted to solid triglycerides (e.g. Crisco) by partial hydrogenation of their unsaturated components. Some of the remaining double bonds are isomerized (to trans) in this operation

Figure 10.12 The shapes of stearic and oleic acids

Saturated Acids

Animal Fats

Unsaturated Acids

3. Waxes

Waxes are esters of fatty acids with long chain monohydric alcohols (one hydroxyl group). Natural waxes are often mixtures of such esters, and may also contain hydrocarbons

Figure 10.13 Waxes are widely distributed in nature. The leaves and fruits of many plants have waxy coatings, which may protect them from dehydration and small predators. The feathers of birds and the fur of some animals have similar coatings which serve as a water repellent. Carnuba wax is valued for its toughness and water resistance.

4. Phospholipids

Phospholipids are the main constituents of cell membranes. They resemble the triglycerides in being ester or amide derivatives of glycerol or sphingosine with fatty acids and phosphoric acid. The phosphate moiety of the resulting phosphatidic acid is further esterified with ethanolamine, choline or serine in the phospholipid itself. The following diagram shows the structures of some of these components.

As ionic amphiphiles, phospholipids aggregate or self-assemble when mixed with water, but in a different manner than the soaps and detergents. Because of the two pendant alkyl chains present in phospholipids and the unusual mixed charges in their head groups, micelle formation is unfavourable relative to a bilayer structure. If a phospholipid is smeared over a small hole in a thin piece of plastic immersed in water, a stable planar bilayer of phospholipid molecules is created at the hole. As shown in the following diagram 10.1, the polar head groups on the faces of the bilayer contact water, and the hydrophobic alkyl chains form

a nonpolar interior. The phospholipid molecules can move about in their half the bilayer, but there is a significant energy barrier preventing migration to the other side of the bilayer.

The bilayer membrane structure is also found in aggregate structures called liposomes. Liposomes are microscopic vesicles consisting of an aqueous core enclosed in one or more phospholipid layers. They are formed when phospholipids are vigorously mixed with water. Unlike micelles, liposomes have both aqueous interiors and exteriors

Figure 10.14 Structure of some important classes of phospholipids

A cell may be considered a very complex liposome. The bilayer membrane that separates the interior of a cell from the surrounding fluids is largely composed of phospholipids, but it incorporates many other

components, such as cholesterol, that contribute to its structural integrity. Protein channels that permit the transport of various kinds of chemical species in and out of the cell are also important components of cell membranes.

The interior of a cell contains a variety of structures (organelles) that conduct chemical operations vital to the cells existence. Molecules bonded to the surfaces of cells serve to identify specific cells and facilitate interaction with external chemical entities.

The sphingomyelins are also membrane lipids. They are the major component of the myelin sheath surrounding nerve fibres. Multiple Sclerosis is a devastating disease in which the myelin sheath is lost, causing eventual paralysis.

1.2.3 Proteins

Proteins are large biological molecules consisting of one or more chains of amino acids. Proteins perform a vast array of functions within living organisms, including catalysing metabolic reactions, replicating DNA, responding to stimuli, and transporting molecules from one location to another. Proteins differ from one

another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in folding of the protein into a specific three-dimensional structure that determines its activity.

A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues.

The sequence of amino acids in a protein is defined by the sequence of a gene, which is encoded in the genetic code.

In general, the genetic code specifies 20 standard amino acids; however, in certain organisms the genetic code can include selenocysteine and in certain archea pyrrolysine. Shortly after or even during synthesis, the residues in a protein are often chemically modified by posttranslational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can also work together to achieve a particular function, and they often associate to form stable protein complexes.

Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in virtually every process within cells. Many proteins are enzymes that catalyse biochemical reactions and are vital to metabolism.

Proteins also have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape. Other proteins are important in cell signalling, immune responses, cell adhesion, and the cell cycle.

Proteins are also necessary in animals' diets, since animals cannot synthesize all the amino acids they need and must obtain essential amino acids from food. Through the process of digestion, animals break down ingested protein into free amino acids that are then used in metabolism. When proteins are digested, amino acids are left. The human body needs a number of amino acids to break down food. Amino acids need to be eaten in large enough amounts for optimal health. Protein foods are no longer described as being "complete proteins" or "incomplete proteins."

Amino acids are found in animal sources such as meats, milk, fish, and eggs, as well as in plant sources such as soy, beans, legumes, nut butters, and some grains (such as wheat germ). You do not need to eat animal products to get all the protein you need in your diet. Amino acids are classified into three groups:

Essential

Nonessential

Conditional

Essential amino acids cannot be made by the body, and must be supplied by food. They do not need to be eaten at one meal. The balance over the whole day is more important. The nine essential amino acids are:

Nonessential amino acids are made by the body from essential amino acids or in the normal breakdown of proteins. They include:

acid

acid

Structure

Most proteins fold into unique 3-dimensional structures. The shape into which a protein naturally folds is known as its native conformation. Although many proteins can fold unassisted, simply through the chemical properties of their amino acids, others require the aid of molecular chaperones to fold into their native states

Primary structure: the amino acid sequence. Secondary structure structures stabilized by hydrogen bonds. The most common examples are the alpha helix, beta sheet and turns. Because secondary structures are local, many regions of different secondary structure can be present in the same protein molecule.

Tertiary structure protein molecule; the spatial relationship of the secondary structures to one another. Tertiary structure is generally stabilized by nonlocal interactions, most commonly the formation of a hydrophobic core, but also through salt bridges, hydrogen bonds posttranslational modifications. The term "tertiary structure" is often used as synonymous with the term fold. The tertiary structure is what controls the basic function of the protein.

Quaternary structure several protein molecules (polypeptide chains), usually called protein subunits in this context, which function as a single protein complex.

Figure 10.16 Protein structure, from primary to quaternary structure.

Proteins are not entirely rigid molecules. In addition to these levels of structure, proteins may shift between several related structures while they perform their functions. The tertiary or quaternary structures are usually referred to as "conformations", and transitions between them are called conformational changes.

Such changes are often induced by the binding of a substrate molecule to an enzyme's active site, or the physical region of the protein that participates in chemical catalysis. In solution proteins also undergo variation in structure through thermal vibration and the collision with other molecules.

Proteins can be informally divided into three main classes, which correlate with typical tertiary structures:

1. globular proteins

2. fibrous proteins

3. Membrane proteins.

Almost all globular proteins are soluble and many are enzymes. Fibrous proteins are often structural, such as collagen, the major component of connective tissue, or keratin, the protein component of hair and nails. Membrane proteins often serve as receptors or provide channels for polar or charged molecules to pass through the cell membrane

A special case of intramolecular hydrogen bonds within proteins, poorly shielded from water attack and hence promoting their own dehydration, are called dehydrons.

Cellular functions

Proteins are the chief actors within the cell, said to be carrying out the duties specified by the information encoded in genes.] With the exception of certain types of RNA, most other biological molecules are relatively inert elements upon which proteins act. Proteins make up half the dry weight of an Escherichia coli cell, whereas other macromolecules such as DNA and RNA make up only 3% and 20%, respectively. The set of proteins expressed in a particular cell or cell type is known as its proteome.

The chief characteristic of proteins that also allows their diverse set of functions is their ability to bind other molecules specifically and tightly.

The region of the protein responsible for binding another molecule is known as the binding site and is often a depression or "pocket" on the molecular surface.

This binding ability is mediated by the tertiary structure of the protein, which defines the binding site pocket, and by the chemical properties of the surrounding amino acids' side chains.

Extremely minor chemical changes such as the addition of a single methyl group to a binding partner can sometimes suffice to nearly eliminate binding

Ex: The aminoacyl tRNA synthetase specific to the amino acid valine discriminates against the very similar side chain of the amino acid isoleucine

Proteins can bind to other proteins as well as to small-molecule substrates. When proteins bind specifically to other copies of the same molecule, they can oligomerize to form fibrils; this process occurs often in structural proteins that consist of globular monomers that self-associate to form rigid fibres

Protein protein interactions also regulate enzymatic activity, control progression through the cell cycle, and allow the assembly of large protein complexes that carry out many closely related reactions with a common biological function. Proteins can also bind to, or even be integrated into, cell membranes. The ability of binding partners to induce conformational changes in proteins allows the construction of enormously complex signalling networks.

Importantly, as interactions between proteins are reversible, and depend heavily on the availability of different groups of partner proteins to form aggregates that are capable to carry out discrete sets of function, study of the interactions between specific proteins is a key to understand important aspects of cellular function, and ultimately the properties that distinguish particular cell types.

Enzymes

The best-known role of proteins in the cell is as enzymes, which catalyse chemical reactions. Enzymes are usually highly specific and accelerate only one or a few chemical reactions. Enzymes carry out most of the reactions involved in metabolism, as well as manipulating DNA in processes such as DNA replication, DNA repair, and transcription. Some enzymes act on other proteins to add or remove chemical groups in a process known as posttranslational modification. About 4,000 reactions are known to be catalysed by enzymes The rate acceleration of as much as 1017-fold increase is provided by enzymes over the uncatalyzed reaction in the case of orotate decarboxylase (78 million years without the enzyme, 18 milliseconds with the enzyme).

The molecules bound and acted upon by enzymes are called substrates.

Although enzymes can consist of hundreds of amino acids, it is usually only a small fraction of the residues that come in contact with the substrate, and an even smaller fraction three to four residues on average that are directly involved in catalysis.



The region of the enzyme that binds the substrate and contains the catalytic residues is known as the active site.

Cell signalling and ligand binding

Many proteins are involved in the process of cell signalling and signal transduction. Some proteins, such as insulin, are extracellular proteins that transmit a signal from the cell in which they were synthesized to other cells in distant tissues. Many receptors have a binding site exposed on the cell surface and an effector domain within the cell, which may have enzymatic activity or may undergo a conformational change detected by other proteins within the cell

Antibodies are protein components of an adaptive immune system whose main function is to bind antigens, or foreign substances in the body, and target them for destruction.



Antibodies can be secreted into the extracellular environment or anchored in the membranes of specialized B cells known as plasma cells.



Whereas enzymes are limited in their binding affinity for their substrates by the necessity of conducting their reaction, antibodies have no such constraints. An antibody's binding affinity to its target is extraordinarily high.



Many ligand transport proteins bind particular small biomolecules and transport them to other locations in the body of a multicellular organism. These proteins must have a high binding affinity when their ligand is present in high concentrations, but must also release the ligand when it is present at low concentrations in the target tissues.



The ligand-binding protein is haemoglobin transports oxygen from the lungs to other organs and tissues in all vertebrates and has close homologs in every biological kingdom

 Lectins are sugar-binding proteins which are highly specific for their sugar moieties. Lectins typically play a role in biological recognition phenomena involving cells and proteins Receptors and hormones are highly specific binding proteins.



Transmembrane proteins can also serve as ligand transport proteins that alter the permeability of the cell membrane to small molecules and ions. The membrane alone has a hydrophobic core through which polar or charged molecules cannot diffuse.



Membrane proteins contain internal channels that allow such molecules to enter and exit the cell. Many ion channel proteins are specialized to select for only a particular ion; for example, potassium and sodium channels often discriminate for only one of the two ions

Structural proteins

Structural proteins confer stiffness and rigidity to fluid biological components. Most structural proteins are fibrous proteins; for example, collagen and elastin are critical components of connective tissue such as cartilage, and keratin is found in hard or filamentous structures such as hair, nails, feathers, hooves, and some animal shells. Some globular proteins can also play structural functions, for example, actin and tubulin are globular and soluble as monomers, but polymerize to form long, stiff fibres that make up the cytoskeleton, which allows the cell to maintain its shape and size. Motor proteins such as myosin, kinesin, and dynein, which are capable of generating mechanical forces. These proteins are crucial for cellular motility of single celled organisms and the sperm of many multicellular organisms which reproduce sexually. They also generate the forces exerted by contracting muscles and play essential roles in intracellular transport.

1.2.4 Nucleic Acids

Nucleic acid, naturally occurring chemical compound that is capable of being broken down to yield phosphoric acid, sugars, and a mixture of organic bases (purines and pyrimidines). Nucleic acids are the main information-carrying molecules of the cell, and, by directing the process of protein synthesis, they determine the inherited characteristics of every living thing. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the master blueprint for life and constitutes the genetic material in all free-living organisms and most viruses. RNA is the genetic material of certain viruses, but it is also found in all living cells, where it plays an important role in certain processes such as the making of proteins.

The first isolation of what we now refer to as DNA was accomplished by Johann Friedrich Miescher circa 1870. He reported finding a weakly acidic substance of unknown function in the nuclei of human white blood cells, and named this material "nuclein". A few years later, Miescher separated nuclein into protein and nucleic acid components. In the 1920's nucleic acids were found to be major components of chromosomes, small

Figure showing the

gene-carrying bodies in the nuclei of complex cells. Elemental analysis of nucleic acids showed the presence of phosphorus, in addition to the usual C, H, N & O. Unlike proteins, nucleic acids contained no sulfur. Complete hydrolysis of chromosomal nucleic acids gave inorganic phosphate, 2-deoxyribose (a previously unknown sugar) and four different heterocyclic bases (shown in the following diagram). To reflect the unusual sugar component, chromosomal nucleic acids are called deoxyribonucleic acids, abbreviated DNA. Analogous nucleic acids in which the sugar component is ribose are termed ribonucleic acids, abbreviated RNA. The acidic character of the nucleic acids was attributed to the phosphoric acid moiety.

Molecular composition

Nucleic acids can vary in size, but are generally very large molecules. Indeed, DNA molecules are probably the largest individual molecules known.

nucleic acid molecules range in size from 21 nucleotides (small interfering RNA) to large chromosomes (human chromosome 1 is a single molecule that contains 247 million base pairs

In most cases, naturally occurring DNA molecules are double-stranded and RNA molecules are single-stranded. There are numerous exceptions, some viruses have genomes made of double-stranded RNA and other viruses have single-stranded DNA genomes, and, in some circumstances, nucleic acid structures with three or four strands can form.

left-deoxyguanosine-depictsthebase,sugar extra aribonucleotide5'and3' ribose(ordeoxyribose)-understandingthisconceptand discussed 3'carbonhasa

Nucleic acids are linear polymers (chains) of nucleotides. Each nucleotide consists of three components: a purine or pyrimidine nucleobases (sometimes termed nitrogenous base or simply base), a pentose sugar, and a phosphate group. The substructure consisting of a

Nucleic acid types differ in the structure of the sugar in their nucleotides - DNA contains 2'deoxyribose while RNA contains ribose (where the only difference is the presence of a hydroxyl group). Also, the nucleobases found in the two nucleic acid types are different: adenine, cytosine, and guanine are found in both RNA and DNA, while thymine occurs in DNA and uracil occurs in RNA.

The sugars and phosphates in nucleic acids are connected to each other in an alternating chain (sugar-phosphate backbone) through phosphodiester linkages. In conventional nomenclature, the carbons to which the phosphate groups attach are the 3'-end and the 5'end carbons of the sugar. This gives nucleic acids directionality, and the ends of nucleic acid molecules are referred to as 5'-end and 3'-end. The nucleobases are joined to the sugars via an N-glycosidic linkage involving a nucleobases ring nitrogen (N-1 for pyrimidines and N-9 for purines) and the 1' carbon of the pentose sugar ring.

Non-standard nucleosides are also found in both RNA and DNA and usually arise from modification of the standard nucleosides within the DNA molecule or the primary (initial) RNA

transcript. Transfer RNA (tRNA) molecules contain a particularly large number of modified nucleosides.

Types of nucleic acids

1. Deoxyribonucleic acid (DNA) Deoxyribonucleic acid is a nucleic acid containing the genetic instructions used in the development and functioning of all known living organisms (with the exception of RNA viruses). The DNA segments carrying this genetic information are called genes. Likewise, other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information. Along with RNA and proteins, DNA is one of the three major macromolecules that are essential for all known forms of life.

 DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds.



These two strands run in opposite directions to each other and are therefore antiparallel. Attached to each sugar is one of four types of molecules called nucleobases (informally, bases).

 It is the sequence of these four nucleobases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins.





Theprocessinvolvingformingphosphodiester bonds nucleotideandthe5'carbon of called "sugarphosphatebackbone",fromwhichthebasesproject.Akeyfeature thattheyhavetwodistinctiveends: the 5'(5tothe 5'and3' DNA(shownabove)andRNA,the5' 3'endahydroxylgroup.Another thatDNAandRNA previously thatnucleicacidsare

Figure 10.21

The two strands of DNA are arranged antiparallel toone another: viewed from left to right the "top" strand is aligned 5' to 3', while the "bottom" strand is aligned 3' to 5'. This is always the casefor duplex nucleic acids. G-C base pairs have 3 hydrogen bonds, whereas A-T base pairs have 2 hydrogen bonds: one consequence of this disparity is that it takes more energy (e.g. a higher temperature) to disrupt GC-rich DNA than AT-rich DNA.

The code is read by copying stretches of DNA into the related nucleic acid RNA in a process called transcription. Within cell DNA is organized into long structures called chromosomes. During cell division these chromosomes are duplicated in the process of DNA replication, providing each cell its own complete set of chromosomes.

Eukaryotic organisms (animals, plants, fungi, and protists) store most of their DNA inside the cell nucleus and some of their DNA in organelles, such as mitochondria or chloroplasts.

 Prokaryotes (bacteria and archea) store their DNA only in the cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide

the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.

2. Ribonucleic acid (RNA)

Ribonucleic acid (RNA) functions in converting genetic information from genes into the amino acid sequences of proteins. The three universal types of RNA include transfer RNA (tRNA), messenger RNA (mRNA), and ribosomal RNA (rRNA).

Messenger RNA acts to carry genetic sequence information between DNA and ribosomes, directing protein synthesis.

Ribosomal RNA is a major component of the ribosome, and catalyses peptide bond formation.

Transfer RNA serves as the carrier molecule for amino acids to be used in protein synthesis, and is responsible for decoding the mRNA. In addition, many other classes of RNA are now known.

Base Pairing and Double Stranded Nucleic Acids

Most DNA exists in the famous form of a double helix, in which two linear strands of DNA are wound around one another. The major force promoting formation of this helix is complementary base pairing: A's form hydrogen bonds with T's (or U's in RNA), and G's form hydrogen bonds with C's. If we mix two ATGC's together, the following duplex will form

1.2.6 Vitamins

Vitamins are substances that your body needs to grow and develop normally. There are 13 vitamins your body needs. They are vitamins A, C, D, E, K and the B vitamins (thiamine, riboflavin, niacin, pantothenic acid, biotin, vitamin B-6, vitamin B-12 and folate). You can usually get all your vitamins from the foods you eat. Your body can also make vitamins D and K. People who eat a vegetarian diet may need to take a vitamin B12 supplement.

Each vitamin has specific jobs. If you have low levels of certain vitamins, you may develop a deficiency disease. For example, if you don't get enough vitamin D, you could develop rickets. Some vitamins may help prevent medical problems. Vitamin A prevents night blindness.



Vitamins are essential for the normal growth and development of a multicellular organism. Using the genetic blueprint inherited from its parents, a foetus begins to develop, at the moment of conception, from the nutrients it absorbs.

It requires certain vitamins and minerals to be present at certain times. These nutrients facilitate the chemical reactions that produce among other things, skin, bone, and muscle. If there is serious deficiency in one or more of these nutrients, a child may develop a deficiency disease. Even minor deficiencies may cause permanent damage

For the most part, vitamins are obtained with food, but a few are obtained by other means. For example, microorganisms in the intestine produce vitamin K and biotin, while one form of vitamin D is synthesized in the skin with the help of the natural ultraviolet wavelength of sunlight. Humans can produce some vitamins from precursors they consume. Examples include vitamin A, produced from beta carotene, and niacin, from the amino acid tryptophan

Once growth and development are completed, vitamins remain essential nutrients for the healthy maintenance of the cells, tissues, and organs that make up a multicellular organism; they also enable a multicellular life form to efficiently use chemical energy provided by food it eats, and to help process the proteins, carbohydrates, and fats required for respiration

Deficiencies

Humans must consume vitamins periodically but with differing schedules, to avoid deficiency.

The human body's stores vitamins A, D, and B12 in significant amounts in the human body, mainly in the liver

An adult human's diet may be deficient in vitamins A and D for many months and B12 in some cases for years, before developing a deficiency condition.

Vitamin B3 (niacin and niacinamide) is not stored in the human body in significant amounts

vitamin C,

first symptoms

scurvy

vitamin

in humans

varied widely, from a month to more than six months, depending on previous dietary history that determined body stores.

of vitamins are classified as either primary or secondary.

primary deficiency occurs

deficiency. In contrast, restrictive diets

potential to cause prolonged vitamin

Vitamin generic descriptor name

Well-known human vitamin deficiencies involve thiamine (beriberi), niacin (pellagra), vitamin C (scurvy), and vitamin D (rickets). In much of the developed world, such deficiencies are rare due to (1) an adequate supply of food and (2) the addition of vitamins and minerals to common foods, often called fortification.

Vitamerche mical name(s)(list not complete)

Retinol,

Solubil ity

Recommended dietary allowances (male,age19 70)

VitaminA

Vitamin

Vitamin

Vitamin

Vitamin

Vitamin

Vitamin

Vitamin

Vitamin

Vitamin

Vitamin

Vitamin

Vitamin

Critical thinking Questions

Deficiencydisease

Upper Intake Level (UL/day)

OverdosediseaseFoodsources

HypervitaminosisA

Orange,ripeyellow fruits,leafy vegetables,carrots, pumpkin,squash, spinach,liver,soy milk,milk

Pork,oatmeal,brown rice,vegetables, potatoes,liver,eggs

Dairyproducts, bananas,popcorn, greenbeans, asparagus

Meat,fish,eggs, manyvegetables, mushrooms,tree

Meat,broccoli, avocados

Meat,vegetables, treenuts,bananas

Raweggyolk,liver, peanuts,certain vegetables

Leafyvegetables, pasta,bread,cereal,

Meatandother animalproducts

Manyfruitsand vegetables,liver

Fish,eggs,liver, mushrooms

Manyfruitsand vegetables,nutsand

Leafygreen vegetablessuchas spinach,eggyolks, liver

1. Why is glycineahighlyconservedaminoacidresidue in theevolution of proteins?

2. Thegeneencodingaproteinwithasingledisulphidebondundergoesamutationthat changesaserineresidueintoacysteineresidue.Proposeadirectmethod to findout whetherthedisulphidepairing in thismutant is thesame as in theoriginalprotein.

3. Theatmosphere of theprimitiveearthbeforetheemergence of lifecontainedN2, NH3, H2,HCN, CO andH2O.Which of thesecompounds is themostlikelyprecursor of most of theatoms in adenine?Why?

4. Areorganicsolventslikebenzeneandetherpolar or non-polarsubstances?

STABILIZING INTERACTIONS

Interactions in nature need not be always Covalent or involving electron sharing

many instances, brief and weak interactions

interactions include:

interactions

bonding

1.3.1 Vander Waals Forces



molecular and atomic level can cause strong stable structures

Van der Waals forces' is a general term used to define the attraction of intermolecular forces between molecules. There are two kinds of Van der Waals forces and stronger dipole-dipole forces

The chance that an electron of an atom is in a certain area in the electron cloud at a specific time is called the "electron charge density." Since there is no way of knowing exactly where the electron is located and since they do not all stay in the same area 100 percent of the time, if the electrons all go to the same area at once, a dipole is formed momentarily.

Even if a molecule is nonpolar, this displacement of electrons causes a nonpolar molecule to become polar for a moment.

Since the molecule is polar, this means that all the electrons are concentrated at one end and the molecule is partially negatively charged on that end.

This negative end makes the surrounding molecules have an instantaneous dipole also, attracting the surrounding molecules' positive ends. This process is known as the London Dispersion Force of attraction.

The ability of a molecule to become polar and displace its electrons is known as the molecule's "Polarizability." The more electrons a molecule contains, the higher its ability to become polar. Polarizability increases in the periodic table from the top of a group to the bottom and from right to left within periods. This is because the higher the molecular mass, the more electrons an atom has.

 With more electrons, the outer electrons are easily displaced because the inner electrons shield the nucleus' positive charge from the outer electrons which would normally keep them close to the nucleus.



When the molecules become polar, the melting and boiling points are raised because it takes more heat and energy to break these bonds. Therefore, the greater the mass, the more electrons present, and the more electrons present, the higher the melting and boiling points of these substances.

 London dispersion forces are stronger in those molecules that are not compact, but long chains of elements. This is because it is easier to displace the electrons because the forces of attraction between the electrons and protons in the nucleus are weaker. The more readily displacement of electrons means the molecule is also more “polarizable.”

Dipole-Dipole Forces



Snapshot Figure 10.23 induced dipole in the approaching molecule, which is orientated in such a way that the + end of one is attracted to the -end of the other.

These forces are similar to London Dispersion forces, but they occur in molecules that are permanently polar versus momentarily polar.

In this type of intermolecular interaction, a polar molecule such as water or H2O attracts the positive end of another polar molecule with its negative end of its dipole. The attraction between these two molecules is the dipole-dipole force.

Van der Waals Equation

Van der Waals equation is required for special cases, such as non-ideal (real) gases, which is used to calculate an actual value. The equation consist of:

(P + n2a / V2)(V − nb) = nRT

Figure 10.24 Whole lattice of molecules could be held together in a solid using van der Waals dispersion forces

The V in the formula refers to the volume of gas, in moles n. The intermolecular forces of attraction is incorporated into the equation with the n

‘a’ is a specific value of a particular gas.

P represents the pressure measured, which is expected to be lower than in usual cases. The variable b expresses the eliminated volume per mole, which accounts for the volume of gas molecules and is also a value of a particular gas.

R is a known constant, 0.08206 L atm mol-1 K-1, and T stands for temperature.

Unlike most equations used for the calculation of real, or ideal, gases, van der Waals equation takes into account, and corrects for, the volume of participating molecules and the intermolecular forces of attraction.

Gecko climbing glass

The ability of geckos using only one toe attributed to the van der Waals forces between these surfaces and the spatula (plural spatulae), or microscopic projections, which cover the hair-like setae found on their footpads. In 2011, a paper was published relating the effect to both velcro-like hairs and the presence of lipids in gecko footprints.

1.3.2 Electrostatic Interactions.

These are interactions between cations and anions, which are functional groups with formal charge. Electrostatic interactions are very strong, and cause the vapour pressure of sodium chloride, for example, to be very low. If you leave table salt out on the counter, how long before it sublimes away?

These interactions, the attractive force between the sodium cation and chloride cation in sodium chloride, are called 'electrostatic interactions'. This name is unfortunate because ALL molecular interactions are inherently electrostatic in nature, so it would be better to avoid the term 'electrostatic interaction' and use the phrase 'charge-charge interaction'. However by convention we call them electrostatic. These interactions are observed between phosphate oxygen of RNA (formal charge -1) and magnesium ions (formal charge +2).

The electrostatic force between two point charges is given by:

Figure1. 25 Orientation of Chargedatoms in NaClcrystal

Force = k q1 q2 / ε r 2 where k = 9.0 x 10 9 nt-meter 2 /coul 2 q = -1.6 x 10 19 coulombs for an electron. r = distance between the point charges (meters) ε = the dielectric constant of the medium (unit less).

ε reflects the tendency of the medium to shield one charge from another. ε is 1 in a vacuum, around 4 in the interior of a protein and 80 in water. The problem of calculating electrostatic effects in proteins is complex in part because of non-uniformity of the dielectric environment. The dielectric micro-environment is variable, with less shielding of charges in regions of hydrocarbon sidechains and greater shielding in regions of polar sidechains.

The electrostatic energy is given by: ΔE= k a q1 q where a = avogadro's number.

One can crudely estimate the energetics of a charge-charge interaction in a protein. The energy of an amine (charge +1) and a carboxylic acid (charge -1) separated by 4 Å in the interior of protein is given by:

ΔE = -(9.0x10 = 87 kjoules/mole = 21 kcal/mole

This rough approximation is around 10-fold greater than the values determined experimentally. Charge-charge interactions fall off slowly with distance (1/r).

1.3.3 Hydrogen Bonding

Hydrogen bonding is an intermolecular or intramolecular attraction that occurs between molecules with hydrogen bond donors and molecules with hydrogen bond acceptors. Hydrogen bond donors are molecules that have a hydrogen attached to an electronegative atom (for example, hydroxyls or amines). Hydrogen bond acceptors are molecules that have a lone pair of electrons located on an electronegative atom (for example, oxygen, nitrogen, or fluorine). Hydrogen bonds are not as strong as covalent and ionic bonds but are stronger than van der Waals interactions. Hydrogen bonding is responsible for the high boiling point of water and is important for the organization of complementary chains of base pairs in DNA and RNA.



A hydrogen atom attached to a relatively electronegative atom is a hydrogen bond donor. This electronegative atom is usually fluorine, oxygen, or nitrogen.



An electronegative atom such as fluorine, oxygen, or nitrogen is a hydrogen bond acceptor, whether it is bonded to a hydrogen atom or not.



An example of a hydrogen bond donor is ethanol, which has a hydrogen bonded to oxygen; an example of a hydrogen bond acceptor which does not have a hydrogen atom bonded to it is the oxygen atom on diethyl ether.

Examples of hydrogen bond donating (donors) and hydrogen bond accepting groups (acceptors)

Cyclic dimer of acetic acid; dashed green lines represent hydrogen bonds

A hydrogen attached to carbon can also participate in hydrogen bonding when the carbon atom is bound to electronegative atoms, as is the case in chloroform, CHCl3. The electronegative atom attracts the electron cloud from around the hydrogen nucleus and, by decentralizing the cloud, leaves the atom with a positive partial charge.

Because of the small size of hydrogen relative to other atoms and molecules, the resulting charge, though only partial, represents a large charge density. A hydrogen bond results when this strong positive charge density attracts a lone pair of electrons on another heteroatom, which becomes the hydrogen-bond Acceptor.

The hydrogen bond is often described as an electrostatic dipole-dipole interaction. It also has some features of covalent bonding: it is directional and strong, produces interatomic distances shorter than sum of van der Waals radii, and usually involves a limited number of interaction partners, which can be interpreted as a type of valence. These covalent features are more substantial when acceptors bind hydrogen from more electronegative donors.

Liquids that display hydrogen bonding are called associated liquids.

Hydrogen bonds can vary in strength from very weak (1–2 kJ mol−1) to extremely strong (161.5 kJ mol−1 in the ion HF−2) Typical enthalpies in vapour include:

1. F−H…: F (161.5 kJ/mol or 38.6 kcal/mol)

2. O−H…: N (29 kJ/mol or 6.9 kcal/mol)

3. O−H…: O (21 kJ/mol or 5.0 kcal/mol)

4. N−H…: N (13 kJ/mol or 3.1 kcal/mol)

5. 6.

Hydrogen bond is most obvious in a water molecule



There are two hydrogen atoms and one oxygen atom. Two molecules of water can form a hydrogen bond between them; the simplest case, when only two molecules are present, is called the water dimer and is often used as a model system.

 When more molecules are present, as is the case with liquid water, more bonds are possible because the oxygen of one water molecule has two lone pairs of electrons, each of which can form a hydrogen bond with a hydrogen on another water molecule.

 This can repeat such that every water molecule is H-bonded with up to four other molecules, as shown in the figure (two through its two lone pairs, and two through its two hydrogen atoms). Hydrogen bonding strongly affects the crystal structure of ice, helping to create an open hexagonal lattice.



The density of ice is less than the density of water at the same temperature; thus, the solid phase of water floats on the liquid, unlike most other substances.



Liquid water's high boiling point is due to the high number of hydrogen bonds each molecule can form, relative to its low molecular mass.



Due to difficulty of breaking these bonds, water has a very high boiling point, melting point, and viscosity compared to otherwise similar liquids not conjoined by hydrogen bonds.



Water is unique because its oxygen atom has two lone pairs and two hydrogen atoms, meaning that the total number of bonds of a water molecule is up to four.



For example, hydrogen fluoride which has three lone pairs on the F atom but only one H atom can form only two bonds; (ammonia has the opposite problem: three hydrogen atoms but only one lone pair).



Because water forms hydrogen bonds with the donors and acceptors on solutes dissolved within it, it inhibits the formation of a hydrogen bond between two molecules of those solutes or the formation of intramolecular hydrogen bonds within those solutes through competition for their donors and acceptors.



Consequently, hydrogen bonds between or within solute molecules dissolved in water are almost always unfavourable relative to hydrogen bonds between water and the donors and acceptors for hydrogen bonds on those solutes

Hydrogen bond: Effects

Dramatically higher boiling points of NH3, H2O, and HF compared to the heavier analogues PH3, H2S, and HCl.

Increase in the melting point, boiling point, solubility, and viscosity of many compounds can be explained by the concept of hydrogen bonding.

Viscosity of anhydrous phosphoric acid and of glycerol

Dimer formation in carboxylic acids and hexamer formation in hydrogen fluoride, which occur even in the gas phase, resulting in gross deviations from the ideal gas law.

Pentamer formation of water and alcohols in apolar solvents.

High water solubility of many compounds such as ammonia is explained by hydrogen bonding with water molecules.

Negative azeotropy of mixtures of HF and water

Deliquescence of NaOH is caused in part by reaction of OH− with moisture to form hydrogen-bonded H3O−

2 species. An analogous process happens between NaNH2 and NH3, and between NaF and HF.

The fact that ice is less dense than liquid water is due to a crystal structure stabilized by hydrogen bonds.

The presence of hydrogen bonds can cause an anomaly in the normal succession of states of matter for certain mixtures of chemical compounds as temperature increases or decreases. These compounds can be liquid until a certain temperature, then solid even as the temperature increases, and finally liquid again as the temperature rises over the "anomaly interval"[29]

Smart rubber utilizes hydrogen bonding as its sole means of bonding, so that it can "heal" when torn, because hydrogen bonding can occur on the fly between two surfaces of the same polymer.

Strength of nylon and cellulose fibres.

Wool, being a protein fibre is held together by hydrogen bonds, causing wool to recoil when stretched. However, washing at high temperatures can permanently break the hydrogen bonds and a garment may permanently lose its shape.

1.3.4 Hydrophobic interactions

Hydrophobic interactions describe the relations between water and hydrophobes (low water-soluble molecules). Hydrophobes are nonpolar molecules and usually have a long chain of carbons that do not interact with water molecules. The mixing of fat and water is a good example of this particular interaction. The common misconception is that water and fat doesn’t mix because the Van der Waals forces that are acting upon both water and fat molecules are too weak. However, this is not the case. The behaviour of a fat droplet in water has more to do with the enthalpy and entropy of the reaction than its intermolecular forces

Causes of Hydrophobic Interactions

American chemist Walter Kauzmann discovered that nonpolar substances like fat molecules tend to clump up together rather than distributing itself in a water medium, because this allow the fat molecules to have minimal contact with water.

Hydrophobic interactions are relatively stronger than other weak intermolecular forces (i.e. Van der Waals interactions or Hydrogen bonds).

Temperature: As temperature increases, the strength of hydrophobic interactions increases also. However, at an extreme temperature, hydrophobic interactions will denature.

Number of carbons on the hydrophobes: Molecules with the greatest number of carbons will have the strongest hydrophobic interactions.

The shape of the hydrophobes: Aliphatic organic molecules have stronger interactions than aromatic compounds. Branches on a carbon chain will reduce the hydrophobic effect of that molecule and linear carbon chain can produce the largest hydrophobic interaction. This is so because carbon branches produce steric hindrance, so it is harder for two hydrophobes to have very close interactions with each other to minimize their contact to water.

Hydrophobic Interactions are important for the folding of proteins. This is important in keeping a protein alive and biologically active, because it allow to the protein to decrease in surface are and reduce the undesirable interactions with water.

Besides from proteins, there are many other biological substances that rely on hydrophobic interactions for its survival and functions, like the phospholipid bilayer membranes in every cell of your body

When a hydrophobe is dropped in an aqueous medium, hydrogen bonds between water molecules will be broken to make room for the hydrophobe; however, water molecules do not react with hydrophobe. This is considered an endothermic reaction, because when bonds are broken heat is put into the system. Water molecules that are distorted by the presence of the hydrophobe will make new hydrogen bonds and form an ice-like cage structure called a clathrate cage around the hydrophobe. This orientation makes the system (hydrophobe) more ordered with an increase of the entropy of the system; therefore ΔS is negative.

The mixing hydrophobes and water molecules is not spontaneous; however, hydrophobic interactions between hydrophobes are spontaneous. When hydrophobes come together and interact with each other, enthalpy increases positive) because some of hydrogen bonds that form the clathrate cage will be broken. Tearing down a portion of the clathrate cage will cause the entropy to increase since forming it decreases the entropy.

According to the formula: ΔG =ΔH−TΔS

ΔH = small positive value

ΔS = large positive value

Water

of 16

10 atoms

Theformation of cagestructure.Cage of water moleculesaroundthenonpolar solute(Hydrophobicentity)

ΔG is negative and hence hydrophobic interactions are spontaneous.

Critical thinking Questions

1. WhydoesPoly-L-leucine, in an organicsolventlikedioxane, is alpha-helicalwhereaspolyL-isoleucine is not?

2. Whattype of non-covalentinteractionsholdstogetherGraphite?

3. AmutationthatchangesAla to Val in theinterior of aproteinleads to loss of activity.A 2nd mutation in differentpositionchangesIsoleucine to glycine.Howdoesthisrestorethe activity?

Hydrophobe

1.4 PRINCIPLES OF BIOPHYSICAL CHEMISTRY

Living cells constantly perform work. They require energy for maintaining their highly organized structures, synthesizing cellular components, generating electric currents, and many other processes.

pH scale is the measure of acidity of a solution. Its value ranges from 0-14. Buffers help to maintain the solution pH by balancing the H+ ion content. Blood is common example of buffer system

Bioenergetics is the quantitative study of energy relationships and energy conversions in biological systems. Biological energy transformations obey the laws of thermodynamics.

All chemical reactions are influenced by two forces: the tendency to achieve the most stable bonding state (for which enthalpy, H, is a useful expression) and the tendency to achieve the highest degree of randomness, expressed as entropy, S. The net driving force in a G, the free-energy change, which represents the net effect of these two factors:

The standard transformed free-energy change, is a physical constant that is characteristic for a given reaction and can be calculated from the equilibrium constant for the reaction: ∆G = ∆RT ln K eq.

The actual free- on the concentrations of reactants and products: G is large and negative, the reaction tends to go G is large and positive, the reaction tends to go in t

The free-energy change for a reaction is independent of the pathway by which the reaction occurs. Free-energy changes are additive; the net chemical reaction that results from successive reactions sharing a common intermediate has an overall free-energy change that is the sum of the G values for the individual reactions.

1.4.1 pH

pH is a measure of the concentration of H+ [H3O+] ions in a solution. Only the concentration of H+ and OHmolecules determine the pH. When the concentration of H+ and OH- ions are equal, the solution is said to be neutral. If there are more H+ than OH- molecules the solution is acidic, and if there are more OH- than H+ molecules, the solution is basic.



Acidic and basic are two extremes that describe a chemical property chemicals.

 Mixing acids and bases can cancel out or neutralize their extreme effects. A substance that is neither acidic nor basic is neutral.



The pH scale measures how acidic or basic a substance is. The pH scale ranges from 0 to 14. A pH of 7 is neutral. A pH less than 7 is acidic. A pH greater than 7 is basic.



The pH scale is logarithmic and as a result, each whole pH value below 7 is ten times more acidic than the next higher value.

For example, pH 4 is ten times more acidic than pH 5 and 100 times (10 times 10) more acidic than pH 6. The same holds true for pH values above 7, each of which is ten times more alkaline (another way to say basic) than the next lower whole value.



For example, pH 10 is ten times more alkaline than pH 9 and 100 times (10 times 10) more alkaline than pH 8.

Pure water is neutral. But when chemicals are mixed with water, the mixture can become either acidic or basic.

 Examples of acidic substances are vinegar and lemon juice. Lye, milk of magnesia, and ammonia are examples of basic substances.



Ionization of Water:

Water molecules exist in equilibrium with hydrogen ions and hydroxide ions. H2O < > H+ + OH-

The water equilibrium constant is written as:

Kw = [H+] [OH-]

Experimentally, it has been found that the concentration of: H+ = OH- = 10-7 : Therefore: Kw = [10-7][ 10-7] = [10-14]

(To multiply exponential numbers - simply add the exponents.)

The values for Kw, H+, and OH- concentration all indicate that the equilibrium favours the reactant (water molecules). In other words, only very small amounts of H+ and OH- ions are present.

Effect of Acids and Bases on Water Equilibrium:

If an acid (H+) is added to the water, the equilibrium shifts to the left and the OH- ion concentration decreases.

Water Equilibrium: H2O <

If base shifts to left and the H+ concentration decreases.

Water Equilibrium Principle: The multiplication product (addition of exponents) of H+ and OH- ion concentration must always be equal to 10-14.

BOTH H+ and OH- ions are ALWAYS PRESENT in any solution. A solution is acidic if the H+ are in excess. A solution is basic, if the OH- ions are in excess.

Definition of pH, pOH, and pKw:

The concentrations of hydrogen ions and indirectly hydroxide ions are given by a pH number. pH is defined as the negative logarithm of the hydrogen ion concentration. The equation is:

pH = - log [H+]

similarly, pOH = - log [OH-

and p Kw = - log [Kw] .

pH Scale:



The pH scale, (0 - 14), is the full set of pH numbers which indicate the concentration of H+ and OHions in water.

The diagram1.30 shows some relationships which summarizes much of the previous discussion. pH Scale Principle: H+ ion concentration and pH relate inversely. OH- ion concentration and pH relate directly. The following statements may be made about the pH scale numbers. a. Increasing pH means the H+ ions are decreasing. b. Decreasing pH means H+ ions are increasing. c. Increasing pH means OH- ions are d. Decreasing pH means OH- ions are



The Strength of an Acid Is Specified by Its Dissociation Constant. a quantity that is a measure of the relative proton affinities of the HA/A- and H3O+/H2O conjugate acid base pairs. Here, as throughout the text, quantities in square brackets symbolize the molar concentrations of the enclosed substances. Since in dilute aqueous solutions the water concentration is essentially constant with [H2O] = 1000 g. L 55.5 M, this term is customarily combined with the dissociation constant, which th

Acids with dissociation constants smaller than that of H3O+ (which, by definition, is unity in aqueous solutions) are only partially ionized in aqueous solutions and are known are weak acids (K < 1). Conversely, strong acids have dissociation constants larger than that of H3O+ so that they are almost completely ionized in aqueous solutions

(for its first ionization), are strong acids. Since strong acids rapidly transfer all their protons to H O, the strongest acid that can stably exist in aqueous solutions is H3O Likewise, there can be no stronger base in aqueous solutions than OH-

(K>1). Many of the so-called mineral acids, such as HClO

The pH of a Solution is determined by the Relative Concentrations of Acids and Bases. The relationship between the pH of a solution and the concentrations of an acid and its conjugate base can be easily derived as Henderson–Hasselbalch equation:

This equation indicates that the pK of an acid is numerically equal to the pH of the solution when the molar concentrations of the acid and its conjugate base are equal.

1.4.2 Buffers

Buffers are an important concept in acid-base chemistry. Here's a look at what buffers are and how they function.



A buffer is an aqueous solution that has a highly stable pH. If you add acid or base to a buffered solution, its pH will not change significantly. Similarly, adding water to a buffer or allowing water to evaporate will not change the pH of a buffer.

A buffer is made by mixing a large volume of a weak acid or weak base together with its conjugate. A weak acid and its conjugate base can remain in solution without neutralizing each other. The same is true for a weak base and its conjugate acid.



When hydrogen ions are added to a buffer, they will be neutralized by the base in the buffer. Hydroxide ions will be neutralized by the acid. These neutralization reactions will not have much effect on the overall pH of the buffer solution.



When you select an acid for a buffer solution, try to choose an acid that has a pKa close to your desired pH. This will give your buffer nearly equivalent amounts of acid and conjugate base so it will be able to neutralize as much H+ and OH- as possible. Refer figure1.31

The curves have similar shapes but are shifted vertically along the pH axis.

The pH at the equivalence point of each titration (where the equivalents of OH- added equal the equivalents of HA initially present) is 7 because of the reaction of A- with H2O to form [HA] = [OH]; similarly, each initial pH is 7. The pH at the midpoint of each titration is numerically equal to the pK of its corresponding acid; here, according to the Henderson

The slope of each titration curve is much less near its midpoint than it is near its wings. This indicates that when [HA] nearly equal to [Athe pH of the solution is relatively insensitive to the addition of strong base or strong acid. Such a solution, which is known as an acid buffer, is resistant to pH changes because small amounts of added H+ or OH-, respectively, react with the A- or HA present without greatly changing the value of log([A]/[HA]).

Buffers Stabilize a Solution’s pH

1-Lsolutions of 1M astrongbase. At the startingpoint of eachtitration, the acidform of the conjugate acid basepairoverwhelminglypredominates. At the midpoint of thetitration,where pH = pK, the concentration of the acid is equal tothat of itsconjugatebase.Finally, at the end point of the titration,where the equivalents of strongbaseadded equal the equivalents of acid at the startingpoint,theconjugate base isin greatexcessoveracid.Theshadedbandsindicate the pH rangesoverwhich the correspondingsolutioncanfunction effectively as abuffer.

The ability of a buffer to resist pH changes with added acid or base is directly proportional to the total concentration of the conjugate acid base pair, [HA] + [A-]. It is maximal when pH = pK and decreases rapidly with a change in pH from that point. A good rule of thumb is that a weak acid is in its useful buffer range within 1 pH unit of its pK (the shaded regions of figure 1.31). Above this range, where

the ratio [A-] / [HA]> 10, the pH of the solution changes rapidly with added strong base. A buffer is similarly impotent with addition of strong acid when its pK exceeds the pH by more than a unit.

4.Thetwo the curve. curvenearitsstartingpointsandendpoints in

Fig.1.31 .This acid and PO3-

Biological fluids, both those found intra cellularly and extra cellularly, are heavily buffered. For example, the pH of the blood in healthy individuals is closely controlled at pH 7.4.The phosphate and carbonate ions that are components of most biological fluids are important in this respect because they have pK’s in this range. Moreover, many biological molecules, such as proteins, nucleic acids, and lipids, as well as numerous small organic molecules, bear multiple acid base groups that are effective as buffer components in the physiological pH range. The concept that the properties of biological molecules vary with the acidity of the solution in which they are dissolved was not fully appreciated before the beginning of the twentieth century so that the acidities of biochemical preparations made before that time were rarely controlled. Consequently these early biochemical experiments yielded poorly reproducible results. More recently, biochemical preparations have been routinely buffered to simulate the properties of naturally occurring biological fluids. In practice, the chosen weak acid and one of its soluble salts are dissolved in the (nearly equal) mole ratio necessary to provide the desired pH and, with the aid of a pH meter, the resulting solution is fine-tuned by titration with strong acid or base

Polyprotic Acids

Substances that bear more than one acid base group, such as H3PO4 or H2CO3, as well as most biomolecules, are known as polyprotic acids.

1.4.3 Reaction Kinetics

Chemical reaction kinetics deals with the rates of chemical processes. Any chemical process may be broken down into a sequence of one or more single-step processes known either as elementary processes, elementary reactions, or elementary steps. Elementary reactions usually involve either a single reactive collision between two molecules, which we refer to as a bimolecular step, or dissociation/isomerisation of a single reactant molecule, which we refer to as a unimolecular step. Very rarely, under conditions of extremely high pressure, a trimolecular step may occur, which involves simultaneous collision of three reactant molecules. An important point to recognise is that many reactions that are written as a single reaction equation in actual fact consist of a series of elementary steps. This will become extremely important as we learn more about the theory of chemical reaction rates.

As a general rule, elementary processes involve a transition between two atomic or molecular states separated by a potential barrier. The potential barrier constitutes the activation energy of the process, and determines the rate at which it occurs. When the barrier is low, the thermal energy of the reactants will generally be high enough to surmount the barrier and move over to products, and the reaction will be fast. However, when the barrier is high, only a few reactants will have sufficient energy, and the reaction will be much slower. The presence of a potential barrier to reaction is also the source of the temperature dependence of reaction rates.

The huge variety of chemical species, types of reaction, and the accompanying potential energy surfaces involved means that the timescale over which chemical reactions occur covers many orders of magnitude, from very slow reactions, such as iron rusting, to extremely fast reactions, such as the electron transfer processes involved in many biological systems or the combustion reactions occurring in flames. A study into the kinetics of a chemical reaction is usually carried out with one or both of two main goals in mind:

1.Analysis of the sequence of elementary steps giving rise to the overall reaction. i.e. The reaction mechanism.

2.Determination of the absolute rate of the reaction and/or its individual elementary steps.

Rate of reaction

When we talk about the rate of a chemical reaction, what we mean is the rate at which reactants are used up, or equivalently the rate at which products are formed. The rate therefore has units of concentration per unit time, mol dm-3 s-1 (for gas phase reactions, alternative units of concentration are often used, usually units of pressure Torr, mbar or Pa). To measure a reaction rate, we simply need to monitor the concentration of one of the reactants or products as a function of time.

There is one slight complication to our definition of the reaction rate so far, which is to do with the stoichiometry of the reaction. The stoichiometry simply refers to the number of moles of each reactant and product appearing in the reaction equation. For example, the reaction equation for the well-known Haber process, used industrially to produce ammonia, is:

N2 + 3H2 

N2 has a stochiometric coefficient of 1, H2 has a coefficient of 3, and NH3 has a coefficient of 2. We could determine the rate of this reaction in any one of three ways, by monitoring the changing 3 concentration of N2, H2, or NH3. For the above reaction, the rate is given by:

Note that a negative sign appears when we define the rate using the concentration of one of the reactants. This is because the rate of change of a reactant is negative (since it is being used up in the reaction), but the reaction rate needs to be a positive quantity.

Rate laws

The rate law is an expression relating the rate of a reaction to the concentrations of the chemical species present, which may include reactants, products, and catalysts. Many reactions follow a simple rate law, which takes the form as given here:

i.e. the rate is proportional to the concentrations of the reactants each raised to some power. The constant of proportionality, k, is called the rate constant. The power a particular concentration is raised to is the order of the reaction with respect to that reactant. Note that the orders do not have to be integers. The sum of the powers is called the overall order. Even reactions that involve multiple elementary steps often obey rate laws of this kind, though in these cases the orders will not necessarily reflect the stoichiometry of the reaction equation.

Multi-step processes may follow simple or complex rate laws, and as the above examples have hopefully illustrated, the rate law generally does not followfrom the overall reaction equation. This makes perfect sense, since the overall reaction equation for a multi-step process is simply the net result of all of the elementary reactions in the mechanism. The ‘reaction’ given in the overall reaction equation never actually takes place! However, even though the rate law for a multi-step reaction cannot immediately be written down from the

reaction equation as it can in the case of an elementary reaction, the rate law is a direct result of the sequence of elementary steps that constitute the reaction mechanism. As such, it provides our best tool for determining an unknown mechanism. Once we know the sequence of elementary steps that constitute the reaction mechanism, we can quite quickly deduce the rate law. Conversely, if we do not know the reaction mechanism, we can carry out experiments to determine the orders with respect to each reactant and then try out various ‘trial’ reaction mechanisms to see which one fits best with the experimental data. At this point it should be emphasised again that for multi-step reactions, the rate law, rate constant, and order are determined by experiment, and the orders are not generally the same as the stoichiometric coefficients in the reaction equation.

A final important point about rate laws is that overall rate laws for a reaction may contain reactant, product and catalyst concentrations, but must not contain concentrations of reactive intermediates

A rate law is a differential equation that describes the rate of change of a reactant (or product) concentration with time. If we integrate the rate law then we obtain an expression for the concentration as a function of time, which is generally the type of data obtained in an experiment. In many simple cases, the rate law may be integrated analytically. Otherwise, numerical (computer-based) techniques may be used. Four of the simplest rate laws are given below in both their differential and integrated form

In the above [A] represent the initial concentrations of A and B i.e. their concentrations at the start of the reaction.

Half lives

The half life, t1/2, of a substance is defined as the time it takes for the concentration of the substance to fall to half of its initial value. Note that it only makes sense to define a half life for a substance not present in excess at the start of the reaction. We can obtain equations for the half-lives for reactions of various orders by substituting the values t = t

1.4.4 Thermodynamics

Thermodynamics is a branch of science, which deals with energy associated with different atoms, molecules and chemical bonds. Thermodynamics helps us to understand the reaction kinetics and driving force for the reactions. Let us examine the basic terminologies of the Thermodynamics, which are required from CSIR point of view.

1. System

Any solution/solid part in which molecules are there and some chemical reactions are happening

E.g.: Solution in a beaker, air filled in balloon, you are a system.

Surrounding

Whatever is present surrounding a system

Air present above the solution

3. Enthalpy ( H)

Greek: enthalpein, to warm in

simple terms, it is the heat content of the system.

does it means? It is the energy stored in every chemical bond in the molecules of the system.

we all know,

form a bond

need to add energy and to break a bond we need to supply energy, when a bond is broken energy is again given out.

If there is a system, called A, whose H = 20, is was heated or some reaction happened, its H change

Now what is the change in Enthalpy? = H2

write it as Change in enthalpy = ∆H = H2 –

U

represents the

is the

is due to random motion of atoms and molecules

which is defined as force times the distance moved under its influence, is associated with organized motion.

Force can be of different forms mass on another,

expansion force exerted by a gas,

the tensional force exerted by a spring or muscle fibre,

the electrical force of one charge on another

Forces of friction and viscosity.

Many chemical reactions release energy in the form of heat, light, or sound. These are exothermic reactions. Exothermic reactions may occur spontaneously and result in higher randomness or entropy (ΔS > 0) of the system. They are denoted by a negative heat flow (heat is lost to the surroundings) and decrease in enthalpy (ΔH < 0). In the lab, exothermic reactions produce heat or may even be explosive.

Figure1.32 You are a system you are surrounded by air , we call it Surrounding exothermic thermitereactionusingiron(III) oxide.Thesparksflying of molten theirwake.

There are other chemical reactions that must absorb energy in order to proceed. These are endothermic reactions. Endothermic reactions cannot occur spontaneously. Work must be done in order to get these reactions to occur. When endothermic reactions absorb energy, a temperature drop is measured during the reaction. Endothermic reactions are characterized by positive heat flow (into the reaction) and an increase in enthalpy (+ΔH).

reaction

of endothermic reactions

decomposition

cation

atom

1.4.3.1 Laws of Thermodynamics

First law of thermodynamics: Energy of the Universe is always constant.

First law of thermodynamics is a mathematical statement of the law of conservation of energy: Energy can be neither created nor destroyed

SECOND LAW OF THERMODYNAMICS:

The Universe Tends Toward Maximum Disorder

Figure 1.34 In the above picture, one boy goes up gaining energy but the other one looses same amount of energy. Thus Total energy of 2

When a swimmer falls into the water (a spontaneous process), the energy of the coherent motion of his body is converted to that of the chaotic thermal motion of the surrounding water molecules. The reverse process, the swimmer being ejected from still water by the sudden coherent motion of the surrounding water molecules, has never been witnessed even though such a phenomenon violates neither the first law of thermodynamics nor Newton’s laws of motion. This is because spontaneous processes are characterized by the conversion of order (in this case the coherent motion of the swimmer’s body) to chaos (here the random thermal motion of the water molecules). The second law of thermodynamics, which expresses this phenomenon, therefore provides a criterion for determining whether a process is spontaneous. Note that thermodynamics says nothing about the rate of a process; that is the purview of chemical

Spontaneity and Disorder

Spontaneous processes occur in directions increase the overall disorder of the universe, that is, of the system and its surroundings. Disorder  Defined as the number of equivalent ways, W, of arranging the components of the universe

Entropy

It is a thermodynamic property which provides a quantitative measure of the disorder of a given thermodynamic state,

Entropy of the system

Figure 1.35 As you can see here, if you throw the bricks in random, most probable is that it will fall randomly as shown right diagram… it will never fall as perfect wall structure. Hence what we can say about it?

is proportional to the number of gas molecules it contains.

Entropy

a state function

depends only on the parameters that describe a state.

The entropy (disorder) of a substance increases with its volume.

entropy depends on concentration.

In some cases

find order is increasing, as in living cell

Living cell tries to attain more and more stability and thus becoming ordered.

it is creating disturbance in outer environment,

releasing some chemicals

using some energy from

Measurement of Entropy

In chemical and biological systems, it is impossible, to determine the entropy of a system by counting the number of ways it can assume its most probable state.

Figure 1.35 In the above fig, the molecules in , Entropy is Entropy is Entropy is higher. In other words, entropy of the system is

For spontaneous processes where is the absolute temperature at which the change in heat occurs.

It is evident, however, that any system becomes progressively disordered (its entropy increases) as its temperature rises

Thus the entropy change of a reversible process at constant temperature can be determined from measurements of the heat transferred and the temperature at which this occurs.

The entropy of the does not change.

Third law of thermodynamics

Entropy of a system becomes 0 as temperature approaches 0 K

The third law of thermodynamics is essentially a statement about the ability to create an absolute temperature scale, for which absolute zero is the point at which the internal energy of a solid is precisely 0.

Various sources show the following three potential formulations of the third law of thermodynamics: It is impossible to reduce any system to absolute zero in a finite series of operations. The entropy of a perfect crystal of an element in its most stable form tends to zero as the temperature approaches absolute zero.

As temperature approaches absolute zero, the entropy of a system approaches a constant.

The third law means a few things, and again all of these formulations result in the same outcome depending upon how much you take into account: However, due to quantum constraints on any physical system, it will collapse into its lowest quantum state but never be able to perfectly reduce to 0 entropy, therefore it is impossible to reduce a physical system to absolute zero in a finite number of steps

Gibbs free energy, G, expresses the amount of energy capable of doing work during a reaction at constant temperature and pressure. When a reaction proceeds with the release of free energy (that is, when the system changes so as to possess less free energy), the free-energy change, ∆G, has a negative value and the reaction is said to be exergonic. In endergonic reactions, the system gains free energy and ∆G is positive.

The units of ∆G and ∆H are joules/mole or calories/mole (recall that 1 cal = 4.184 J); units of entropy are Joules /mole. Kelvin (J/mol- K) (Table 1.6). Under the conditions existing in biological systems (including constant temperature and pressure), changes in free energy, enthalpy, and entropy are related to each other quantitatively by the equation. ∆G=∆H - T∆S.

In which ∆G is the change in Gibbs free energy of the reacting system, ∆H is the change in enthalpy of the system, T is the absolute temperature, and ∆S is the change in entropy of the system. By convention, ∆S has a positive sign when entropy increases and ∆H, as noted above, has a negative sign when heat is released by the system to its surroundings. Either of these conditions, which are typical of favorable processes, tend to make ∆G negative. In fact, ∆G of a spontaneously reacting system is always negative. The second law of thermodynamics states that the entropy of the universe increases during all chemical and physical processes, but it does not require that the entropy increase take place in the reacting system itself. The order produced within cells as they grow and divide is more than compensated for by the disorder they create in their surroundings in the course of growth and division. In short, living organisms preserve their internal order by taking from the surroundings free energy in the form of nutrients or sunlight, and returning to their surroundings an equal amount of energy as heat and entropy.

The composition of a reacting system (a mixture of chemical reactants and products) tends to continue changing until equilibrium is reached. At the equilibrium concentration of reactants and products, the rates of the forward and reverse reactions are exactly equal and no further net change occurs in the system. The concentrations of reactants and products at equilibrium define the equilibrium constant, Keq. In the general reaction aA + bB cC + dD, where a, b, c, and d are the number of molecules of A, B, C, and D participating, the equilibrium constant is given by

Where [A], [B], [C], and [D] are the molar concentrations of the reaction components at the point of equilibrium.

Under standard conditions (298 K = 25 C), when reactants and products are initially present at 1 M concentrations or, for gases, at partial pressures of 101.3 kilopascals (kPa), or 1 atm, the force driving the system toward equilibrium is defined as the standard free- definition, the standard state for reactions that involve hydrogen ions is [H+] = 1 M, or pH 0. Most biochemical reactions, however, occur in well-buffered aqueous solutions near pH 7; both the pH and the concentration of water (55.5 M) are essentially constant. For convenience of calculations, biochemists therefore define a different standard state, in which the concentration of H+ is 10 M (pH 7) and that of water is 55.5 M; for reactions that involve Mg2+ (including most in which ATP is a reactant), its concentration in solution is commonly taken to be constant at 1 mM. Physical constants based on this biochemical standard state are called standard transformed constants and are written with a prime (such as ∆G’ transformed constants used by chemists and physicists. By convention, when H O, H+, and/or Mg2+ are reactants or products, their concentrations are incorporated into the constants K’eq and ∆G’

Just as K eq is a physical constant characteristic for each reaction, so too is ∆G a constant. There is a simple relationship between K eq and ∆G’

The standard free-energy change of a chemical reaction is simply an alternative mathematical way of expressing its equilibrium constant. If the equilibrium constant for a given chemical reaction is 1.0, the standard free-energy change of that reaction is 0.0 (the natural logarithm of 1.0 is zero). If K’eq of a reaction is greater than 1.0, its ∆G is negative. If K’eq is less than 1.0, ∆G is positive. Because the relationship between ∆G and K’eq is exponential, relatively small changes in ∆G correspond to large changes in K’eq.

Table 1.6 Relationships among Keq, G, and the Direction of Chemical Reactions under Standard Conditions

Sample problem

Calculatethethestandardfree-energychange of thereactioncatalysed by theenzyme phosphoglucomutase:Glucose1-phosphate  glucose6-phosphate

Solution:Chemicalanalysisshowsthatwhether we startwith, say, 20 mM glucose1-phosphate (but no glucose6-phosphate) or with 20 mM glucose6-phosphate (but no glucose1-phosphate),the final equilibriummixture at25oCand pH 7.0will be thesame:1 mM glucose1-phosphateand 19 mM glucose 6-phosphate.(Rememberthatenzymes donot affect the point of equilibrium of areaction;theymerely hastenitsattainment.)Fromthesedata we can calculate the equilibriumconstant:

Becausethestandardfree-energychange negative,whenthereactionstartswith1.0Mglucose1phosphateand1.0Mglucose6-phosphate, glucose6phosphateproceedswithaloss(release)

1.4.5 Colligative properties

Solutions have different properties than either the solutes or the solvent used to make the solution. Those properties can be divided into two main groups colligative properties. Colligative properties depend only on the number of dissolved particles in solution and not on their identity. Non colligative properties d

To explain the difference between the two sets of solution properties, we will compare the properties of a 1.0

Despite the concentration of sodium chloride being half of the sucrose concentration, both solutions have precisely the same number of dissolved particles because each sodium chloride unit creates two particles upon dissolution

Therefore, any difference in the properties of those two solutions is due to a non colligative property.

Both solutions have the same freezing point, boiling point, pressure, and osmotic pressure because those colligative properties of a solution only depend on the number of dissolved particles. The taste of the two solutions, however, is markedly different.

The sugar solution is sweet and the salt solution tastes salty. Therefore, the taste of the solution is not a colligative pr M solution of CuSO salt and sugar solutions. Other non-colligative properties include viscosity, surface tension, and solubility.

Raoult's Law and Vapour Pressure Lowering

When a non volatile solute is added to a liquid to form a solution, the vapour pressure above that solution decreases. To understand why that might occur, let's analyse the vaporization process of the pure solvent then do the same for a solution. Liquid molecules at the surface of a liquid can escape to the gas phase when they have a sufficient amount of energy to break free of the liquid's intermolecular forces. That vaporization process is reversible. Gaseous molecules coming into contact with the surface of a liquid can be trapped by intermolecular forces in the liquid. Eventually the rate of escape will equal the rate of capture to establish a constant, equilibrium vapour pressure above the pure liquid.

 If we add a non-volatile solute to that liquid, the amount of surface area available for the escaping solvent molecules is reduced because some of that area is occupied by solute particles.

Therefore, the solvent molecules will have a lower probability to escape the solution than the pure solvent.

That fact is reflected in the lower vapour pressure for a solution relative to the pure solvent. That statement is only true if the solvent is non-volatile. If the solute has its own vapour pressure, then the vapour pressure of the solution may be greater than the vapour pressure of the solvent.

Note that we did not need to identify the nature of the solvent or the solute (except for its lack of volatility) to derive that the vapour pressure should be lower for a solution relative to the pure solvent. That is what makes vapour pressure lowering a colligative property it only depends on the number of dissolved solute particles.

The French chemist Francois Raoult discovered the law that mathematically describes the vapour pressure lowering phenomenon. Raoult's law is given in:

Raoult's law states that the equals the mole fraction of the solvent, c vapour pressure of the pure solvent, P While that "law" is approximately obeyed by most solutions, some show deviations from the expected Deviations from Raoult's law can either be positive or negative. A positive deviation means that there is a higher than expected vapour pressure above the solution. A negative deviation, conversely, means that we find a lower than expected pressure for the solution.

The reason for the deviation stems from a flaw in our consideration of the that the solute did not interact with the solvent at all. That, of course, is not true most of the time. If the solute is strongly held by the solvent, then the solution will show a negative deviation from Raoult's law escape from solution.

If the solute and solvent are not as tightly bound to each other as they are to themselves, then the solution will show a positive deviation from Raoult's law because the solvent molecules will find it easier to escape from solution into the gas phase.

Solutions that obey Raoult's law are called ideal solutions because they behave exactly as we would predict. Solutions that show a deviation from Raoult's law are called non ions because they deviate from the expected . Very few solutions actually approach ideality, but Raoult's law for the ideal solution is a good enough approximation for the non ideal solutions that we will continue to use Raoult's law. Raoult's law is the starting point for most of our discussions about the rest of the colligative properties, as we shall see in the

Boiling Point Elevation

One consequence of Raoult's law is that the boiling point of a solution made of a liquid solvent with a non volatile solute is greater than the boiling point of the pure solvent. The boiling point of a liquid or is defined as the temperature at which the vapour pressure of that liquid equals the atmospheric pressure. For a solution, the vapour pressure of the solvent is lower at any given temperature. Therefore, a higher temperature is required to boil the solution than the pure solvent..

As you can see in the figure 1.37 the vapour pressure of the solution is lower than that of the pure solvent. Because both pure solvent and solution need to reach the same pressure to boil, the solution requires a higher temperature to boil. If we represent the difference in boiling point between the pure solvent and a solution as ΔTb, we can calculate that change in boiling point from the:

In this we use the units molality, m, for the concentration, m, because molality is temperature independent. The term Kb is a boiling point elevation constant that depends on the particular solvent being used. The term i in the above equation is called the van't Hoff factor and represents the number of dissociated moles of particles per mole of solute. The van't Hoff factor is 1 for all non electrolyte solutes and equals the total

TheVapourPressure of a of thePure the pure left)thereare the surface right-handsolutionflask. morelikelythatsolvent gasphase the right. solutionshouldhavea lowervaporpressurethanthepure

number of ions released for electrolytes. Therefore, the value of i for Na2SO4 is 3 because that salt releases three moles of ions per mole of the salt.

Freezing Point Depression

As you may have noticed when we looked at the , the freezing point is depressed due to the vapour pressure lowering phenomenon. The points out that fact:

In analogy to the boiling point elevation, we can calculate the amount of the freezing point

depression with the :

Note that the sign of the change in freezing point is negative because the freezing point of the solution is less than that of the pure solvent. Just as we did for boiling point elevation, we use molality to measure the concentration of the solute because it is temperature independent. Do not forget about the van't Hoff factor, in your freezing point calculations.

One way to rationalize the freezing point depression phenomenon without talking about Raoult's law is to consider the freezing process. In order for a liquid to freeze it must achieve a very ordered state that results in t crystal. If there are impurities in the liquid, i.e. solutes, the liquid is inherently less ordered. Therefore, a solution is more difficult to freeze than the pure solvent so a lower temperature is required to freeze the liquid.

Osmotic Pressure

Figure 1.38 Phase Diagram for a Solution and the Pure Solvent Indicating the Freezing Point Depression

Osmosis refers to the flow of solvent molecules past a semipermeable membrane that stops the flow of solute molecules only. When a solution and the pure solvent used in making that solution are placed on either side of a semipermeable membrane, it is found that more solvent molecules flow out of the pure solvent side of the membrane than solvent flows into the pure solvent from the solution side of the membrane. That flow of solvent from the pure solvent side makes the volume of the solution rise. When the height difference between the two sides becomes large enough, the net flow through the membrane ceases due to the extra pressure exerted by the excess height of the solution chamber. Converting that height of solvent into units of pressure gives a measure of the osmotic pressure exerted on the solution by the pure solvent. P stands for pressure, r is the density of the solution, and h is the height of the solution.

You can understand why more molecules flow from the solvent chamber to the solution chamber in analogy to our discussion of Raoult's law. More solvent molecules are at the membrane interface on the solvent side of the membrane than on the solution side. Therefore, it is more likely that a solvent molecule will pass from the solvent side to the solution side than vice versa. That difference in flow rate causes the solution volume to rise. As the solution rises, by the pressure depth equation, it exerts a larger pressure on the membrane's surface. As that pressure rises, it forces more so flow from the solution side to the solvent side. When the flow from both sides of the membrane are equal, the solution height stops rising and remains at a height reflecting the osmotic pressure of the solution.

Figure 1.39 shows a typical setup for measuring the osmotic

The equation relating the osmotic pressure of a solution to its concentration has a form quite similar to the ideal gas law:

Although the above equation may more useful. This form of the equation has been derived by realizing that n / V gives the concentration of the solute in units of molarity, M.

Critical thinking Questions

1. What

2. What reason?

3. What

1.5.1 Bioenergetics

Snapshot

1. Living cells constantly perform work. They require energy for maintaining their highly organized structures, synthesizing cellular components, generating electric currents, and many other processes.

2. Bioenergetics is the quantitative study of energy relationships and energy conversions in biological systems. Biological energy transformations obey the laws of thermodynamics.

3. ATP is the chemical link between catabolism and anabolism. It is the energy currency of the living cell. The exergonic conversion of ATP to ADP and Pi, or to AMP and PPi, is coupled to many endergonic reactions and processes.

4. Direct hydrolysis of ATP is the source of energy in the conformational changes that produce muscle contraction but, in general, it is not ATP hydrolysis but the transfer of a phosphoryl, pyrophosphoryl, or adenylyl group from ATP to a substrate or enzyme molecule that couples the energy of ATP breakdown to endergonic transformations of substrates.

5. Through these group transfer reactions, ATP provides the energy for anabolic reactions, including the synthesis of informational molecules and for the transport of molecules and ions across membranes against concentration gradients

6. Cells contain other metabolites with large, negative, free energies of hydrolysis, including phosphoenolpyruvate, 1,3phosphoglycerate, and phosphocreatine. These high-energy compounds, like ATP, have a high phosphoryl group transfer potential; they are good donors of the phosphoryl group. Thioesters also have high free energies of hydrolysis.

7. Inorganic polyphosphate, present in all cells, may serve as a reservoir of phosphoryl groups with high group transfer

Basis of Thermodynamics



Every living cell and organism must perform work to stay alive, to grow and to reproduce. The ability to harvest energy from nutrients or photons of light and to channel it into biological work is the miracle of life.



Living organisms carry out a remarkable variety of energy transductions. The biological energy transductions obey the physical laws that govern all natural processes, including the laws of thermodynamics.

1st Law of Thermodynamics

The energy of the universe remains constant.

2nd Law of Thermodynamics

All spontaneous processes increase the entropy of the universe.

State functions depend only on the initial and final conditions not on path taken between the initial and final conditions. They are independent of path. The important state functions for the study of biological systems are:

G, the Gibbs free energy which is equal to the total amount of energy capable of doing work during a process at constant temperature and pressure.



If ∆G is negative, then the process is spontaneous and termed exergonic.



If ∆G is positive, then the process is nonspontaneous and termed endergonic.

If ∆G is equal to zero, then the process has reached equibrium. H, the Enthalpy which is the heat content of the system.





When ∆H is negative the process produces heat and is termed exothermic.

When ∆H is positive the process absorbs heat and is termed endothermic.

S, the Entropy is a quantitative expression of the degree of randomness or disorder of the system.

When ∆S is positive then the disorder of the system has increased.

When ∆S is negative then the disorder of the system has decreased.

The conditions of biological systems are constant temperature and pressure. Under such conditions the relationships between the change in free energy, enthalpy and entropy can be described by the expression where T is the temperature of the system in Kelvin.

∆G = ∆H − T∆S

Equilibrium Constants

All spontaneous processes proceed until equilibrium is reached. Consider the following chemical reaction.

The forward rate of product formation is = k

The reverse rate of reactant formation is = k

At equilibrium the concentrations of products and reactants are such that forward and reverse rates are equal

A little algebra and presto

At equilibrium ∆G = 0.

The biochemist standard state the concentration of reactants and products are initially set at 1 M, the temperature is 298°K, the pressure is 1 atm, the pH is 7.0 and the concentration of water is 55 M. The biochemists constants are written as ∆G°’ and K’eq. This is the only standard state we will work with in this class so forgive if I occasionally drop the prime. ∆G°’ is a constant characteristic for each reaction just as K’eq is a constant characteristic for each reaction. These two constants have a simple relationship.

The actual free energy change depends on the reactant and product concentrations.

Reactions can be coupled together. The standard free energy changes are additive. Cool feature of state functions. Multiply the equilibrium constants

Thermodynamics of ATP Hydrolysis (Figure 1.5.1)

ATP is the principle energy currency of the cell that links catabolism to anabolism. ATP has a large negative standard free energy change of hydrolysis.

What is the chemical basis of the large, negative free energy change?

1. The hydrolytic cleavage of the γ-phosphate anhydride bond relieves electrostatic repulsion in ATP. The phosphate formed is stabilized by several resonance forms that are not possible in ATP.

2. The ADP product immediately ionizes, releasing H in a medium with low hydrogen ion concentration, pH 7.

3. ATP has a small solvation energy compared to the solvation energies of ADP, Pi and H+.

4. Thus the products of hydrolysis are stabilized more by solvation than then reactant ATP.

ATP hydrolysis

ATP4- + H2O

∆G°’ = -30.5 kJ/mol

In cells, the concentration of ATP, ADP and Pi are not 1M. For example in human erythrocytes the concentration of ATP is 2.25 mM, [ADP] = 0.25 mM and [Pi] = 1.65 mM.

This ∆G for ATP hydrolysis in the cell is designated ∆Gp. For intact cells, ∆Gp ranges from -50 to -65 kJ/mol. This is known as the phosphorylation potential.

Other High Energy Phosphorylated compounds.

Understanding Biochem

Thegenomes of chimpanzees and humansare 99.9%identical,yetthedifferences betweenthetwospeciesarevast.Therelativelyfewdifferences in genetic endowmentmustexplainthe possession of language by humans,theextraordinary athleticism of chimpanzees, and myriadotherdifferences.Genomiccomparisonwill allowresearchers to identifycandidate genes linked to divergences in the developmentalprograms of humans and theotherprimates and to theemergence of complexfunctionssuch as language.Thepicturewillbecomecleareronly as more primategenomesbecomeavailableforcomparisonwiththehumangenome.

Understanding Biochem

Thehighlyspecialized leaves (Dionaeamuscipula foldtogether alighttouch unsuspecting entrappingtheinsectfor laterdigestion.Attracted nectar theinsecttouchesthree mechanicallysensitive hairs,triggeringthetraplike closing movement sudden changes in mesophyllcells(the innercells probablyachieved release cellsandtheresulting efflux, by Digestiveglands leaf’s enzymesthatextract nutrientsfromtheinsect.

Figure1.5.1.Chemicalbasisforthelargefree-energychange associatedwithATPhydrolysis.1Thechargeseparationthatresults fromhydrolysisrelieveselectrostaticrepulsionamongthefour ATP.2Theproductinorganicphosphate(Pi) is whicheach of the of doublebondcharacter and thehydrogenion is notpermanentlyassociated with any one of theoxygens.(Somedegree of resonance stabilizationalsooccurs in phosphatesinvolved in ester or anhydride linkages,butfewerresonanceformsarepossiblethanfor Pi.)3The productADP2immediatelyionizes,releasingaprotonintoamedium of verylow [H](pH 7).Afourthfactor(notshown)thatfavorsATP hydrolysis is thegreaterdegree of solvation(hydration) of the products Pi and ADPrelative to ATP,whichfurtherstabilizesthe productsrelative to thereactants.

Figure1.5.2. thisreaction is followed in PEP, and thustheproducts

• Phosphoenolpyruvate contains one phosphate ester bond that can under go hydrolysis to yield the enol form of pyruvate which immediately tautomerizes to the more stable keto form of pyruvate.

• The reactant PEP has only one stable form while the product pyruvate has two possible forms. This extra stabilization of the product is the greatest contributor to the high standard free energy of hydrolysis.

1,3-Bisphosphoglycerate

1,3-Bisphosphoglycerate -49.3 kJ/mol

• This high energy compound contains one phosphoanhydride bond

Figure1.5.3. Hydrolysis of 1,3-bisphosphoglycerate.Thedirectproduct of hydrolysis is 3-phosphoglycericacid, with an undissociatedcarboxylicacidgroup, but dissociationoccursimmediately.Thisionizationandtheresonance structures it makespossiblestabilizetheproductrelative to thereactants.Resonancestabilization of Pi further contributes to thenegativefreeenergychange.

• The product 3- phosphoglyceric acid immediately ionizes to produce a carboxylate anion.

• The removal of the 3-phosphoglyceric acid and the resonance stabilized phosphate favor the forward reaction.

Phosphocreatine (Figure 1.5.4)

Phosphocreatine2- + H2 OCreatine + Pi 2- ∆G°’ = -49.3 kJ/mol

Figure1.5.4.Hydrolysis phosphocreatineproduces creatine,which alsoresonance stabilized.

• The release of Pi and the resonance stabilized creatine favor the forward reaction.

• Note for all of these phosphate releasing reactions, the phosphate formed is stabilized by resonance favoring product formation.

The compounds with more negative free energy of phosphate ester hydrolysis than ATP can phosphorylate ADP to form ATP. Ie. PEP + ADP  ATP + pyruvate ∆G°’ = -31.4 kJ/mol. ATP provides energy by group transfers, not simple hydrolysis.

Another example

Glutamate + NH3 + ATP Glutamine +ADP + P

• Most of the group transfer reactions of ATP are SN2 nucleophilic substitutions.

• In the examples above the nucleophile is an oxygen of an alcohol.

• Each of the three phosphates of ATP are susceptible to nucleophilic attack.

• Nucleophilic attack at theγ-phosphate results in ADP and the transfer of phosphate to the nucleophile. Nucleophilic attack at the β-phosphate results in AMP and the transfer of a pyrophosphate group to the nucleophile.

• Nucleophilic attack at the α-phosphate results in pyrophosphate and the adenylylation of the nucleophile.

Figure1.5.5.ATPhydrolysis in twosteps.(a)Thecontribution of ATP to areaction is oftenshown as asinglestep,but is almost alwaysatwo-stepprocess.(b)Shownhere is thereactioncatalyzed by ATPdependentglutaminesynthetase.1Aphosphorylgroup is transferredfromATP to glutamate,then2thephosphorylgroup is displaced by NH3 and released as Pi.

Inorganic Pyrophosphatase -phosphate of ATP results in an adenylylated nucleophile and

The ubiquitous enzyme inorganic pyrophosphatase provides an additional thermodynamic push for the adenylylation reaction by catalyzing the hydrolysis of pyrophosphate into two

Palmitate + ATP + CoASH palmitoyl-CoA + AMP + 2 Pi ∆G° ’ = −65.7 kJ/mol +31.4 kJ/mol= −34.3 kJ/mol

Transphosphorylation

• There are other nucleoside triphosphates (GTP, CTP, UTP, dATP, dGTP, dCTP and dTTP). These are all energetically equivalent to ATP.

• These nucleotides are generated and maintained by phosphoryl group transfer to the corresponding nucleoside diphosphates and monophosphates.

• ATP is the primary high energy nucleoside produced by catabolism. Several enzymes catalyse the transfer of the phosphoryl group from ATP to the other nucleotides.

• There are called nucleoside diphosphate kinases

Note this reaction is also fully reversible, so that the enzyme can also convert AMP to ADP when AMP and ATP concentrations are high. There is a similar enzyme four guanosine nucleotides, Guanylate kinase. Phosphocreatine (PCr) serves as a ready source of phosphoryl groups for quick synthesis of ATP from ADP.

• It is found in the skeletal muscle at a concentration 10 times greater than the cellular concentration of ATP.

• It is also found in smooth muscle, the brain and the kidney at lower concentration. Creatine Kinase catalyzes the following reaction.

When a sudden demand for energy depletes the concentration of ATP, the PCr reservoir is used to replenish ATP at a much faster rate than ATP can be synthesizes by catabolic pathways. When the demand for ATP diminishes, ATP synthesized by catabolism replenishes

High Energy Thioesters Thioesters have a large negative free energy of hydrolysis. As an

1. The metabolism of carbohydrates is dominated by glucose because this sugar is an important fuel molecule in most organisms. If cellular energy reserves are low, glucose is degraded by the glycolytic pathway. Glucose molecules that are not required for immediate energy production are stored as either glycogen (in animals) or starch (in plants).

2. During glycolysis, glucose is phosphorylated and cleaved to form two molecules of glyceraldehyde-3-phosphate. Each ceraldehyde-3-phosphate is then converted to a molecule of pyruvate. A small amount of energy is captured in two molecules each of ATP and NADH. In anaerobic organisms, pyruvate is converted to waste products. During this process, NAD_ is regenerated so that glycolysis can continue. In the presence of O2, aerobic organisms convert pyruvate to acetyl-CoA and then to CO2 and H2O. Glycolysis is controlled primarily by allosteric regulation of three

3. During gluconeogenesis, molecules of glucose are synthesized from noncarbohydrate precursors (lactate, pyruvate,

4. is largely the reverse of glycolysis. The three irreversible glycolytic reactions (the synthesis of pyruvate, the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate, and the formation of glucose from glucose-6-phosphate) are

5. The pentose phosphate pathway, in which glucose-6-phosphate is oxidized, occurs in two phases. In the oxidative phase, two molecules of NADPH are produced as glucose-6- phosphate is converted to ribulose-5-phosphate. In the nonoxidative phase, ribose-5-phosphate and other sugars are synthesized. If cells need more NADPH than ribose-5phosphate, a component of nucleotides and the nucleic acids, then metabolites of the nonoxidative phase are converted into glycolytic intermediates.

6. Several sugars other than glucose are important in vertebrate carbohydrate metabolism. These include fructose, galactose, and mannose.

7. The substrate for glycogen synthesis is UDP-glucose, an activated form of the sugar. UDP-glucose pyrophosphorylase catalyses the formation of UDP-glucose from glucose-1-phosphate and UTP. Glucose-6-phosphate is converted to glucose-1-phosphate by phosphoglucomutase. Glycogen synthesis requires two enzymes: glycogen synthase and branching enzyme. Glycogen degradation requires glycogen phosphorylase and debranching enzyme. The balance between glycogenesis (glycogen synthesis) and glycogenolysis (glycogen breakdown) is carefully regulated by several hormones (insulin, glucagon, and epinephrine) and allosteric regulators.

D-Glucose is a major fuel for most organisms. D-Glucose metabolism occupies the centre position for all metabolic pathways. Glucose contains a great deal of potential energy. The complete oxidation of Glucose yields −2,840 kJ/mol of energy.

Glucose + 6O2 6CO2 + 6H2 O ∆G °’ = −2,840 kJ/mol

Glucose also provides metabolic intermediates for biosynthetic reactions. Bacteria can use the skeletal carbon atoms obtained from glucose to synthesize every amino acid, nucleotide, cofactor and fatty acid required for life. For higher plants and animals there are three major metabolic fates for glucose.

Nearly every living cell catabolizes glucose and other simple sugars by a process called glycolysis. Glycolysis differs from one species to another only in the details of regulation and the fate of pyruvate. Glycolysis is the metabolic pathway that catabolizes glucose into two molecules of pyruvate.

Glycolysis occurs in the cytosol of cells and is essentially an anaerobic process since the pathway’s principle steps do not require oxygen. The glycolytic pathway is often referred to as the in honour

glucoseutilization.

Althoughnottheonlypossiblefatesforglucose,these in terms of the in mostcells.

pioneers who discovered it. The glycolytic pathway is shown below. Glycolysis, which consists of 10 reactions, occurs in two stages:

1. Glucose is phosphorylated twice and cleaved to form two molecules of glyceraldehyde-3phosphate (G-3-P). The two ATP molecules consumed during this stage are like an investment, because this stage creates the actual substrates for oxidation in a form that is trapped inside the

2. Glyceraldehyde-3-phosphate is converted to pyruvate. Four ATP and two NADH molecules are produced. Because two ATP were consumed in stage 1, the net production of ATP per glucose

The glycolytic pathway can be summed up in the following equation:

The Reactions of the Glycolytic Pathway

The 10 reactions of the glycolytic pathway are as follows.

Understanding Biochem

Conversion of galactose1-phosphate to glucose1-phosphate involvestwonucleotide derivatives: UDP-galactose and UDP-glucose.Geneticdefects in any of thethreeenzymes thatcatalyseconversion of galactose to glucose1-phosphate result in galactosemias of varyingseverity.

1. Synthesis of glucose-6-phosphate.

Immediately after entering a cell, glucose and other sugar molecules are phosphorylated. Phosphorylation prevents transport of glucose out of the cell and increases the reactivity of the oxygen in the resulting phosphate ester.

Several enzymes, called the hexokinases, catalyze the phosphorylation of hexoses in all cells in the body. ATP, a cosubstrate in the reaction, is complexed with Mg2+.(ATP-Mg2+ complexes are common in kinase-catalyzed reactions.)

Under intracellular conditions the reaction is irreversible; that is, the enzyme has no ability to retain or accommodate the product of the reaction in its active site, regardless of the concentration of G-6-P.

2. Conversion of glucose-6-

During reaction 2 of glycolysis, the open chain form of the aldose glucose-6-phosphate is converted to the open chain form of the ketose fructose-6-phosphate byphosphoglucose isomerase (PGI) in a readily reversible reaction:

3. The phosphorylation of fructose-6Phosphofructokinase-1 (PFK-1) irreversibly catalyzes the phosphorylation of fructose-6-phosphate to form fructose-1,6-bisphosphate:

The PFK-1-catalyzed reaction is irreversible under cellular conditions. It is, therefore, the first committed step in glycolysis.

Unlike glucose-6- phosphate and fructose-6-phosphate, the substrate and product, respectively, of the previous reaction, fructose-1,6-bisphosphate cannot be diverted into other pathways.

Investing a second molecule of ATP serves several purposes.

First of all, because ATP is used as the phosphorylating agent, the reaction proceeds with a large decrease in free energy.

After fructose-1,6-bisphosphate has been synthesized, the cell is committed to glycolysis.

fructose-1,6-bisphosphate eventually splits into two trioses, another purpose for phosphorylation is to prevent any later product from diffusing out of the cell because charged molecules cannot easily cross membranes.

4. Cleavage of fructose-1,6-bisphosphate.

Stage 1 of glycolysis ends with the cleavage of fructose-1,6-bisphosphate into two three-carbon molecules: glyceraldehyde-3-phosphate (G-3-P) and dihydroxyacetone phosphate (DHAP).

This reaction is an aldol cleavage, hence the name of the enzyme: aldolase.

Although the cleavage of fructose-1,6-bisphosphate is thermodynamically unfavorable, the

5. The interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.

Of the two products of the aldolase reaction, only G-3-P serves as a substrate for the next reaction

To prevent the loss of the other three-carbon unit from the glycolytic pathway, triose phosphate

After this reaction, the original molecule of glucose has been converted to two molecules of G-3-P.

6. Oxidation of glyceraldehyde-3-

During reaction 6 of glycolysis, G-3-P undergoes oxidation and phosphorylation. The product, glycerate-1,3-bisphosphate, contains a high-energy phosphoanhydride bond, which may be used in the next reaction to generate ATP:

This complex process is catalyzed by glyceraldehyde-3-phosphate dehydrogenase, a tetramer composed of four identical subunits.

Each subunit contains one binding site for G-3-P and another for NAD+, an oxidizing ogent. As the enzyme forms a covalent thioester bond with the substrate, a hydride ion (H: _) is transferred to NAD+ in the active site.

NADH, the reduced form of NAD+, then leaves the active site and is replaced by an incoming NAD+ . The acyl enzyme adduct is attacked by inorganic phosphate and the product leaves the active site.

7. Phosphoryl group transfer.

In this reaction ATP is synthesized as phosphoglycerate kinase catalyzes the transfer of the highenergy phosphoryl group of glycerate-1,3-bisphosphate to ADP:

Reaction 7 is an example of a substrate-level phosphorylation. Because the synthesis of ATP is endergonic, it requires an energy source. In substratelevel phosphorylations, ATP is produced by the transfer of a phosphoryl group from a substrate with a high phosphoryl transfer potential (glycerate1,3-bisphosphate) to produce a compound with a lower transfer potential (ATP).

Because two molecules of glycerate-1,3-bisphosphate are formed for every glucose molecule, this reaction produces two ATP molecules, and the investment of phosphate bond energy is recovered. ATP synthesis later in the pathway represents a net gain.

8. The interconversion of 3-

Glycerate-3-phosphate has a low phosphoryl group transfer potential. As such, it is a poor candidate for further ATP synthesis.

Cells convert glycerate-3-phosphate with its energy-poor phosphate ester to phosphoenolpyruvate (PEP), which has an exceptionally high phosphoryl group transfer potential.

In the first step in this conversion (reaction 8), phosphoglycerate mutase catalyzes the conversion of a C-3 phosphorylated compound to a C-2 phosphorylated compound through a two-step addition/elimination cycle.

9. Dehydration of 2-phosphoglycerate.

Enolase catalyzes the dehydration of glycerate-2-phosphate to form PEP:

PEP has a higher phosphoryl group transfer potential than does glycerate-

2- phosphate because it contains an enol-phosphate group instead of a simple phosphate ester. The reason for this difference is made apparent in the next reaction.

Aldehydes and ketones have two isomeric forms. The enol form contains a carbon-carbon double bond and a hydroxyl group.

Enols exist in equilibrium with the more stable carbonyl-containing keto form. The interconversion of keto and enol forms, also called tautomers, is referred to as

This tautomerization is restricted by the presence of the phosphate group, as is the resonance stabilization of the free phosphate ion. As a result, phosphoryl transfer to ADP in reaction 10 is highly favoured.

10. Synthesis of pyruvate.

In the final reaction of glycolysis, pyruvate kinase catalyzes the transfer of a phosphoryl group from PEP to ADP. Two molecules of ATP are formed for each molecule of glucose.

PEP is irreversibly converted to pyruvate because in this reaction the transfer of a phosphoryl group from a molecule with a high transfer potential to one with a lower transfer potential there is an exceptionally large free energy loss.

This energy loss is associated with the spontaneous conversion (tautomerization) of the enol form of pyruvate to the more stable keto form. The 10 reactions of glycolysis are illustrated in (Figure 1.5.8). Glycolysis consists of 10 enzyme catalyzed reactions.

The pathway can be broken down into two phases.

The first phase encompasses the first five reactions to the point that glucose is broken down into 2 molecules of glyceraldehyde 3-phosphate.

Phase 1 consumes two molecules of ATP.

The second phase includes the last five reactions converting glyceraldehyde 3-phosphate into pyruvate. Phase 2 produces 4 molecules of ATP and 2 molecules of NADH.

The net reaction (Phase 1 + Phase 2) produces 2 molecules of ATP and 2 molecules of NADH per molecule of glucose.

9 of the ten metabolites of glycolysis are phosphorylated. Phosphorylated intermediates serve 3 functions.

1. The phosphoryl groups are ionized at physiological pH giving them a net negative electrostatic charge. Biological membranes are impermeable to charged molecules. Intermediates are held within the cell.

2. The transfer of phosphoryl groups conserves metabolic energy. The energy released in breaking the phosphoanhydride bonds of ATP is partially conserved in the formation of phosphate esters.

3. High-energy phosphate compounds formed in glycolysis donate phosphoryl groups to ADP to form ATP.

4. The enzymes of glycolysis use the binding energy of phosphate groups to lower the activation energy and increase the specificity of the enzyme reactions.

1. Chemiosmotic theory provides the intellectual framework for understanding many biological energy transductions, including oxidative phosphorylation and photophosphorylation. The mechanism of energy coupling is similar in both cases: the energy of electron flow is conserved by the concomitant pumping of protons across the membrane, producing

2. In mitochondria, hydride ions removed from substrates by NAD-linked dehydrogenases donate electrons to the

3. Shuttle systems convey reducing equivalents from cytosolic NADH to mitochondrial NADH. Reducing equivalents from

4. Reducing equivalents are then passed through a series of Fe-S centers to ubiquinone, which transfers the electrons to cytochrome b, the first carrier in Complex III. In this complex, electrons take two separate paths through two b-type cytochromes and cytochrome c1 to an Fe-S center. The Fe-S center passes electrons, one at a time, through and into Complex IV, cytochrome oxidase. This copper-containing enzyme, which also contains

5. Some electrons enter this chain of carriers through alternative paths. Succinate is oxidized by succinate dehydrogenase (Complex II), which contains a flavoprotein that passes electrons through several Fe-S centers to ubiquinone. Electrons

6. Plants also have an alternative, cyanide-resistant NADH oxidation pathway. The flow of electrons through Complexes I, III, and IV results in pumping of protons across the inner mitochondrial membrane, making thematrix alkaline relative to the intermembrane space. This proton gradient provides the energy (in the form of the proton-motive force) for ATP synthesis from ADP and Pi by ATP synthase (FoF1 complex) in the inner membrane. ATP synthase carries out which the flow of protons through Fo causeseach of three nucleotide-binding sites in F1 to cycle

7. ATP formation on the enzyme requires little energy; the role of the proton-motive force isto push ATP from its binding site on the synthase.

The complete oxidation of glucose by molecular oxygen is:

6 H12O6 + 6O2 → 6CO2 + 6H2O ΔG°′ = -2823 kJ/mol

This equation is broken down into two half-reactions.

The glucose carbon atoms are oxidized by water molecules:

C6 H12O6 + 6H62O → 6CO2 + 24H+ + 24e- {Glycolysis and citric acid cycle}

The molecular oxygen is reduced by the protons and electrons produced by glucose oxidation:

2 + 24H+ + 24e-  12H2 O {Electron transport and oxidation}

The oxidation of glucose carbon atoms is carried out in glycolysis and the citric acid cycle,and the produced protons and electrons are stored in NADH and FADH2 molecules.

The NADH and FADH2 formed in glycolysis, fatty acid oxidation, and the citric acid cycle are energy-rich molecules because each contains a pair of electrons having a high transfer potential.

When these electrons are used to reduce molecular oxygen to water, a large amount of free energy is liberated, which can be used to generate ATP.

Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH 2 to O 2 by a series of electron carriers.

This process, which takes place in mitochondria, is the major source of ATP in aerobic organisms.

For example, oxidative phosphorylation generates 26 of the 30 molecules of ATP that are formed when glucose is completely oxidized to CO2 and H2O.

The Mitochondrion

Is located inside of cells.

Size: Ellipsoid of ~0.5 μm diameter and ~1 μm length.

Inner compartment called matrix contains:

Soluble enzymes for oxidative metabolism.

substrates

nucleotide

cofactors

inorganic salts

DNA, RNA, ribosomes

Outer membrane:

carbohydrates in cellularrespiration.Stage1:oxidation of fatty someaminoacidsyieldsacetyl-CoA.Stage2: thecitricacidcycleincludesfour whichelectronsareabstracted.Stage3:electronscarried mitochondrial bound)electroncarriers the H2O.Thiselectron

Has porins which have non-specific pores that permit free diffusion up to 10 kD molecules.

Thus the concentration of ions in the intermembrane space and cytosol are nearly the same.

Inner membrane:

- is ~75% protein.

Is impermeable to most hydrophilic substances, freely permeable only to O2, CO2 and H2O.

Contains respiratory chain proteins and transport proteins that control [ATP], [ADP],[pyruvate], [Ca2+], [Pi], and etc.

Generates ionic gradients between cytosol and mitochondria.

Oxidative Phosphorylation

In the electron transport chain, 10NADH+10H

2FADH

Both reactions are highly exergonic (NADH oxidation has a ΔG° of FADH

Oxidative phosphorylation combines the oxidation of NADH or FADH to produce ATP.

Energy captured from the exergonic oxidation reactions is stored in the terminal phosphate group of ATP.

Substrate-level phosphorylation captures energy directly from highly-energetic substrates and stores it in ATP.

Oxidative phosphorylation takes place in the mitochondria, and specifically in the inner (semipermeable) membrane, or matrix membrane.

Electron Transport Chain

The electron transport chain (ETC) begins on the matrix side of the inner mitochondrial membrane.

amitochondrion.

Cytosolic NADH can only enter into oxidative phosphorylation as FADH produced by the malateaspartate shuttle.

Complex IV of the electron transport chain receives electrons from cytochrome C (from Complex III ← CoQ ← Complex I ← NADH), and utilizes oxygen as the final electron acceptor, reducing it to water.

The -52.6kcal/mol of energy liberated by NADH oxidation is released slowly by the electron transport chain, rather than at any single point, and immediately stored by the production of a hydrogen gradient by pumping protons from the matrix to the intermembrane space (and hence, to the cytoplasm).

Complex I pumps four protons, Complex III pumps two protons, and Complex IV pumps four protons, resulting in ten protons pumped per electron pair (NADH molecule) entering the chain.

Because of this proton gradient, matrix pH is much higher than cytosolic pH (8.4 versus 7.4, or a tenfold difference in proton concentration).

A membrane potential across the inner mitochondrial membrane is set up due to the mass moving of positive charges out of the matrix, negatively charging the matrix space, resulting in an electrochemical proton gradient (or "proton-motive force").

Oxidative phosphorylation is the culmination of a series of energy transformations that are called cellular respiration or simply respiration in their entirety.

First, carbon fuels are oxidized in the citric acid cycle to yield electrons with high transfer potential. Then, this electron-motive force is converted into a proton-motive force and, finally, the proton-motive force is converted into phosphoryl transfer potential.

The conversion of electron-motive force into proton-motive force is carried out by three electron-driven proton pumps NADH-Q oxidoreductase, Q-cytochrome c oxidoreductase, and cytochrome c oxidase.

These large transmembrane complexes contain multiple oxidation-reduction centers, including quinones, flavins, iron-sulfur clusters, hemes, and copper ions.

The final phase of oxidative phosphorylation is carried out by ATP synthase, an ATPsynthesizing assembly that is driven by the flow of protons back into the mitochondrial matrix.

Components of this remarkable enzyme rotate as part of its catalytic mechanism. Oxidative phosphorylation vividly shows that protongradients are an interconvertible currency of free energy in biological systems.

Succinate and FADH2

Succinate as an electron donor enters the ETC through Complex II (succinate dehydrogenase), passing its electrons via Complex II to CoQ (coenzyme Q).

FADH

Both succinate and FADH pump only six protons up the gradient, since they bypass Complex I, which pumps four protons.

ATP Synthesis

To capture the energy resident in the proton gradient, adenine nucleotide translocase (ANT) exchanges cytosolic ADP for matrix ATP, making ATP available to the cell and ADP available to Complex V (ATP Synthase), which phosphorylates ADP using energy captured from proton influx through its central channel.

ANT dissipates the proton gradient by one proton per nucleotide pair exchanged.

While Complex V only requires the transport of three protons for synthesis of each ATP, ANT requires the transport of one additional proton to make that ATP available to the cell, resulting in a net use of four protons from the gradient for each ATP produced. Thus oxidation of NADH results in the production of 2.5 ATP, and oxidation of FADH Chemiosmotic Theory

Forty years ago, the inner mitochondrial membrane was suggested to be semipermeable. In particular, it was thought to be impermeable to protons.

The ETC pumps protons out of the matrix, setting up a proton gradient across the matrix membrane.

Energy from the oxidation of electron carriers is converted to the energy of the chemiosmotic gradient.

The chemiosmostic theory received the Nobel prize when it was proven correct.

Complex V Blockers

Complex V blockers dramatically increase the magnitude of the proton-motive force, since they block ATP-synthase-driven dissipation of the gradient.

This stops ATP synthesis and shuts down the ETC, since it does not have enough energy to pump protons up such a steep gradient.

NADH oxidation will be shut down since NADH will accumulate, oxygen will not be used, and will then accumulate to toxic levels.

Proton Gradient Uncouplers

Proton gradient uncouplers shut down ATP synthesis by providing alternate paths for gradient dissipation which do not produce ATP.

Complex V is "coupled" to the electron transport chain, since the ETC requires Complex V to dissipate the proton gradient it produces.

Proton channels and proton shuttles uncouple ATP synthase from the ETC.

oxidation and ETC activity continues, since the ETC doesn't care if ATP is synthesized or not. In fact, NADH oxidation is stimulated, since the proton gradient is completely broken down, and there is very little resistance to proton transport.

Oxygen utilization is increased when NADH oxidation increases.

Figure1.5.

to mitochondria,electronsfromNADH carriers arrangedasymmetrically

themembrane,producingbothachemicalgradient(pH)

specificchannels(Fo).Theproton-motiveforcethatdrivesprotonsbackintothematrixprovidesthe energyforATPsynthesis,catalyzed

Dinitrophenol is an uncoupler, since it is a membrane-soluble proton acceptor, so it can act as a proton shuttle, facilitating the movement of protons down the gradient.

Uncouplers increase temperature, since the energy released is no longer stored as chemical energy, but is simply released as heat.

Thermogenin, a human protein involved in thermoregulation, is a carefully-controlled uncoupler which redirects ETC-produced energy to heat production from ATP production.

Malate-aspartate shuttle

- is the mammalian system.

is more energy efficient than glycerophosphate shuttle.

1.The cytosolic NADH reduces oxaloacetate to malate.

2.The malate is transported into the matrix through a malate-α-ketoglutarate carrier (antiport).

Figure1.5.13.Malate-aspartateshuttle.ThisshuttlefortransportingreducingequivalentsfromcytosolicNADHinto themitochondrialmatrix thecytosol(intermembranespace)passes tworeducingequivalents oxaloacetate,producingmalate.2Malatecrossestheinnermembraneviathemalate ketoglutaratetransporter.3 theresultingNADH is oxidized by therespiratorychain.Theoxaloacetateformedfrommalatecannotpassdirectlyintothecytosol.4 It is firsttransaminated aspartate,which5canleaveviatheglutamate-aspartatetransporter.6Oxaloacetate is regenerated

3.The transported malate reduces the NAD+ in the matrix to NADH, and becomes oxaloacetate.

4.The oxaloacetate receives an amino group from a Glu, and becomes Asp. The deaminated Glu becomes a α-ketoglutarate.

5.Asp in the matrix is transported into the cytosol through a glutamate-aspartate carrier (antiport).

6.The transported Asp donates its amino group to α ketoglutarate, and Asp becomes oxaloacetate, and α ketoglutarate becomes Glu.

1.5.4. Coupled Reaction

A spontaneous reaction may drive a non-spontaneous reaction.

Free energy changes of coupled reactions are additive

Examples of different types of coupling:

A. Some enzyme-catalyzed reactions are interpretable as two coupled half-reactions, one spontaneous and the other non-spontaneous. At the enzyme active site, the coupled reaction is kinetically facilitated,

while the individual half-reactions are prevented. The free energy changes of the half-reactions may be summed, to yield the free energy of the coupled reaction.

For example, in the reaction catalyzed by the Glycolysis enzyme Hexokinase, the two half-reactions are:

ATP + H2O  ADP + Pi DGo' = -31 kJoules/mol

Pi + glucose  glucose-6-P + H2O ... DGo' = +14 kJoules/mol Coupled reaction:

ATP + glucose  ADP + glucose-6-P.. DGo' = -17 kJoules/mol

The structure of the enzyme active site, from which water is excluded, prevents the individual hydrolytic reactions, while favoring the coupled reaction.

B. Two separate enzyme-catalyzed reactions occurring in the same cellular compartment, one spontaneous and the other non-spontaneous may be coupled by a common intermediate

A hypothetical, but typical, example involving

Overall: A + ATP + H

Pyrophosphate ) is often the product of a reaction that needs a driving force. Its spontaneous hydrolysis, catalyzed by Pyrophosphatase enzyme, drives the reaction for which PP is a product.

For an example of such a reaction, see the discussion of cAMP formation below.

o

o The reaction is highly spontaneous due to the production of

C. Ion transport synthesis of ATP.

It should be recalled that the ATP hydrolysis/synthesis reaction is

The free energy change (electrochemical potential difference) across a membrane from side 1 to side 2 is represented

R = gas constant, T = temperature, Z = charge on the ion, F = Faraday constant, and DY = voltage across the membrane.

Since free energy changes are additive, the spontaneous direction for the coupled reaction will depend on the relative magnitudes of:DG for the ion flux (DG varies with the ion gradient and voltage.)DG for the chemical reaction (DGo' is negative in the direction of ATP hydrolysis. The magnitude of DG depends also on concentrations of ATP, ADP, and Pi .)

Two examples of such coupling are:

1. Active transport. Spontaneous ATP hydrolysis (negative DG) is coupled to (drives) ion flux against a gradient (positive DG). For an example, see the discussion of SERCA.

2. ATP synthesis in mitochondria. Spontaneous H+ flux across a membrane (negative DG) is coupled to (drives) ATP synthesis (positive DG). See the discussion of the ATP Synthase.

1.5.5. Group Transfer

High energy" bonds are often represented by the " ~" symbol (squiggle), with ~P representing a phosphate group with a high free energy of hydrolysis.

Compounds with "high energy" bonds are said to have high group transfer potential. For example, Pi may be spontaneously removed from ATP for transfer to another compound (e.g., to a hydroxyl group on glucose).

Potentially two "high energy" bonds can be cleaved, as two phosphates are released by hydrolysis from ATP (adenosine triphosphate), yielding ADP (adenosine diphosphate), and ultimately AMP (adenosine monophosphate). This may be represented as follows (omitting waters of hydrolysis):

ATP often serves as an energy source. Hydrolytic cleavage of one or both of the "high energy" bonds of ATP is coupled to an energy-requiring (non-spontaneous) reaction, as in the examples presented above.

AMP functions as an energy sensor and regulator of metabolism. When ATP production does not keep up with needs, a higher portion of a cell's adenine nucleotide pool is in the form of AMP. AMP then stimulates metabolic pathways that produce ATP.

Some examples of this role involve direct allosteric activation of pathway enzymes by AMP. (E.g., activation of the Glycogen Phosphorylase enzyme by AMP will be discussed later.)

Some regulatory effects of AMP are mediated by the enzyme AMP-Activated Protein Kinase.

1.5.6. Biological Energy Transducers

Energy is found in various forms

Potential energy is energy that is stored in some manner

Most stored energy in biological systems is stored chemically, i.e., within chemical bonds

Organisms are energy transducers, entities that transform energy from one form into another

Organisms are energy transducers

Organisms are transducers of energy (and thereby are less than 100% efficient) who employ the energy they've harnessed to grow, repair, and maintain their bodies, compete with other organisms, and to produce new organisms (babies)

In the process of doing these things, organisms generate waste chemicals and heat.

Organisms create local regions of order at the expense of using up some fraction of the total supply of useful energy found in the universe (but don't fret too much, the energy would have been used up anyway)

When electrons flow from a low affinity carrier (e.g., glucose) to a high affinity carrier (e.g., O2), an electromotive force (emf) will be generated (with energy released and work done).

Energy transducers (proteins) are needed.

Oxidation of energy-rich biological fuels often means dehydrogenation (catalyzed by dehydrogenases) from carbons having various oxidation states.

In the living cells, electrons are transferred directly as electrons (between metal ions), as hydrogen atoms (H++e ), or as a hydride ion (:H or H++2e ).

1. Almost every biochemical reaction is catalyzed by an enzyme. With the exception of a few catalytic RNAs, all known enzymes are proteins.

2. Many require nonprotein coenzymes or cofactors for their catalytic function.

3. Enzymes are classified according to the type of reaction they catalyze. All enzymes have formal E.C. numbers and names, and most have trivial names.

4. Enzymes are highly effective catalysts, commonly enhancing reaction rates by a factor of 10

5. Enzyme-catalyzed reactions involve formation of a complex between substrate and enzyme (an ES complex). Substrate binding occurs in a pocket on the enzyme called the active site.

6. Enzymes lower the activation energy of a reaction and thereby enhance the reaction rate. The equilibrium of a reaction is unaffected by the enzyme.

7. A significant part of the energy used for enzymatic rate enhancements is derived from weak interactions hydrogen bonds and hydrophobic and ionic interactions) between substrate and enzyme. Binding energy also accounts for the exquisite specificity of enzymes for their substrates.

8. Additional catalytic mechanisms employed by enzymes include general acid-base catalysis, covalent catalysis, and metal ion catalysis.

9. Catalysis often involves transient covalent interactions between the substrate and the enzyme, or group transfers to and from the enzyme, so as to provide a new, lower-energy reaction path.

10. Most enzymes have certain kinetic properties in common. When substrate is added to an enzyme, the reaction rapidly achieves a steady state in which the rate at which the ES complex forms balances the rate at which it reacts. As [S] increases, the steady-state activity of a fixed concentration of enzyme increases in a hyperbolic fashion to approach a characteristic maximum rate, Vmax, at which essentially the entire enzyme has formed a complex with substrate.

11. Km is the substrate concentration that results in a reaction rate equal to one-half Vmax, which is characteristic for each enzyme acting on a given substrate.

12. Reversible inhibition of an enzyme is competitive, uncompetitive, or mixed.

13. Competitive inhibitors compete with substrate by binding reversibly to the active site, but they are not transformed by the enzyme.

14. Uncompetitive inhibitors bind only to the ES complex, at a site distinct from the active site.

15. Mixed inhibitors bind to either E or ES, again at a site distinct from the active site.

16. In irreversible inhibition an inhibitor binds permanently to an active site by forming a covalent bond or a very stable noncovalent interaction.

17. The activities of metabolic pathways in cells are regulated by control of the activities of certain enzymes.

18. In feedback inhibition, the end product of a pathway inhibits the first enzyme of that pathway.

19. The activity of allosteric enzymes is adjusted by reversible binding of a specific modulator to a regulatory site.

20. Modulators may be the substrate itself or some other metabolite, and the effect of the modulator may be inhibitory or stimulatory.

21. The kinetic behavior of allosteric enzymes reflects cooperative interactions among enzyme subunits.

22. Other regulatory enzymes are modulated by covalent modification of a specific functional group necessary for activity.

23. The phosphorylation of specific amino acid residues is a particularly common way to regulate enzyme activity.

24. Many proteolytic enzymes are synthesized as inactive precursors called zymogens, which are activated by cleavage of small peptide fragments.assembly.

Properties of enzymes

catalytic

degree of

substrates

chemical reactions

in aqueous conditions

Nature of Enzymes

Most of the Enzymes

proteins

or dissociation

Enzyme

catalytic RNA i.e. Ribozymes).

to loss of catalytic function.

of protein enzymes are essential to their catalytic activity.

secondary,

have molecular weights

enzymes require cofactor.

from about 12,000 to more than 1 million.

can be either inorganic ions (eg. Fe2+, Mg2+, Mn2+ or Zn2+) or can be a complex of organic or metallo-organic molecule called a coenzyme.

group - A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme protein.

A complete, catalytically active enzyme together with its bound coenzyme and/or metal ions.

Apoprotein / Apoenzyme

Function of Coenzyme

Enzyme Classification

Because of many ambiguities in the enzyme names, and the ever-increasing number of newly discovered enzymes, biochemists, by international agreement, have adopted a system for naming and classifying enzymes. This system divides enzymes into six classes, each with subclasses, based on the type of reaction catalyzed.

name,

name

name Hexokinase) and its E.C.

Enzyme Catalysis

Under biological conditions, uncatalyzed reactions tend to be slow because most biomolecules are quite stable in the environment inside the cells.

An enzyme provides a specific environment within which a given reaction can occur more rapidly. Some important terminologies

Active site - catalytic site of an enzyme

Substrate - molecule bound in the active site & acted upon by the enzyme

Ground state - starting point for forward or reverse reaction

Reaction intermediate - any species on the reaction pathway

has a transient existence

and EP complexes]

Rate-limiting step - step (or steps) with the highest activation energy (slow step)

A simple Enzymatic reaction is represented in the following way:

E = Enzyme

S = Substrate

P = Product

ES = Enzyme-Substrate complex (transient)

EP = Enzyme-Product complex (transient)

An Enzyme increases the rate of the reaction, but does not affect the reaction equilibrium.

The reaction coordinate diagram represents the energy changes associated with the progress of th reaction S

The free energy of the system is plotted against the progress of the reaction (the reaction coordinate).

Ground state is the starting point for either the forward or the reverse directions.

The free energy change occurring in standard set of conditions (temperature 298 K; partial pressure of each gas 1 atm, or 101.3 kPa; concentration of each solute 1 M) is standard free energy change ΔG°.

Biochemical standard freedefined as the standard free-energy change at pH 7.0.

In the above reaction coordinate diagram, the free energy of the ground state of P is lower than that of S, i.e. ΔG’° = negative, i.e. the equilibrium favors formation of P.

If ΔG’° = positive, then equilibrium favors formation of S.

of thesystem thereaction

Even if ΔG’° is negative, it does not mean that S →P conversion will occur at a detectable rate.

The rate of reaction does not depend on the position of the equilibrium.

The energy barrier between S and P is the energy required for alignment of reacting groups, formation of transient unstable charges, bond rearrangements, and other transformations required for the reaction to proceed in either direction.

Molecules must overcome this barrier to proceed with the reaction.

Transition state: The top of the energy hill where decay to the S or P state is equally probable. (Note: Transition state is not an intermediate species, not to be confused with ES or EP)



Activation energy (ΔG‡): The difference between the energy levels of the ground state and the transition state.

Higher activation energy corresponds to a slower reaction, and vice versa.

So, to lower the activation energy, a catalyst is added.

Catalysts enhance reaction rates by lowering activation energies. (Figure 1.6.2.)

Enzymes accelerate the interconversion of S and P. The enzyme is not used up in the process, and the equilibrium point is unaffected. However, with the use of enzymes, the equilibrium is reached faster.

In the reaction coordinate diagram comparing an enzyme uncatalyzed and a catalyzed reaction, the ES and EP intermediates occupy minimum energy.

ΔG‡cat is the activation energy for an enzyme catalyzed reaction and is lower than ΔG‡uncat which is the activation energyfor an enzyme uncatalyzed reaction.

Reaction intermediate: any species on the reaction pathway that has a finite chemical lifetime (longer than a molecular vibration, ~10 seconds).

In the above reaction coordinate diagram, ES and EP can be considered as reaction intermediates.

The interconversion of two sequential reaction intermediates thus constitutes a reaction step. In a multi-step reaction, the overall rate of reaction is determined by the step/s with the highest activation energy.

This step/s is the Rate Limiting Step.

Equilibrium constant (K’ interconversion at equilibrium is,

Figure1.6.2.Reactioncoordinatediagram comparingenzymecatalyzedanduncatalyzed ,the ES and EP theenergy theenzyme-catalyzedreaction. correspond to the activationenergyfortheuncatalyzedreactionand theoverallactivationenergyforthecatalyzed activationenergy is lowerwhentheenzymecatalyzesthereaction. Visit:http://study.biotecnika.orgforcoloredpicture.

From thermodynamics, the relationship between K’eq and ΔG’° can be described by the expression

where R = gas constant, 8.315 J/mol*K

T = absolute temperature, 298 K (25 °C)

The equilibrium constant is directly related to the overall standard free energy change for the reaction. A large negative value for ΔG’° reflects favorable reaction equilibrium (but does not mean rate of reaction is high).

For a unimolecular reaction, S P, the rate (or velocity) of reaction (V) represents the amount of S that reacts per unit time.

This is expressed by the rate equation:

Where k = rate constant, [S] = concentration of Substrate

This is a first order reaction as rate of reaction is only dependent on the concentration of substrate. k has units of reciprocal time (eg. s-1).

If a reaction rate depends on the concentration of two different compounds, or if the reaction is between two molecules of the same compound, the reaction is second order and k is a second-order rate constant, with units of M-1s-1. The rate equation then becomes



The expression that relates the magnitude of a rate constant to the activation energy:

where k = Boltzmann constant and h = Planck’s constant.



The relationship between the rate constant k and the activation energy ΔG‡ is inverse and exponential (i.e. a lower activation energy has a faster reaction rate).

Enzyme catalytic power and specificity



The rate enhancements brought about by catalysts are in the range of 5 to 17 orders of magnitude. Enzymes can lower the activation energy partly by rearrangement of covalent bonds during an enzymecatalyzed reaction.



Catalytic functional groups on an enzyme may form a transient covalent bond with a substrate and activate it for reaction, or a group may be transiently transferred from the substrate to the enzyme in the active site of enzyme.

 Enzymes can lower the activation energy partly by weak, noncovalent interactions between enzyme and substrate.



The interactions that stabilize a protein structure are the same ones that are involved in interaction between E and S to form ES complex.

 Formation of each weak interaction in the ES complex is accompanied by release of a small amount of free energy that provides a degree of stability to the interaction.

Binding Energy (ΔG



Binding energy is a major source of free energy used by enzymes to lower the activation energies.



The enzyme active site is not complementary to the substrate, but to the transition states through which substrates pass as they are converted to products.



Consider a hypothetical reaction, in which a metal stick (S) is to be broken into 2 pieces (P). Before the stick is broken, it must first be bent (the transition state). In both stickase examples, magnetic interactions take the place of weak bonding interactions between enzyme and substrate. (Figure 1.6.3.)



An imaginary enzyme (stickase) is designed to catalyze breakage of a metal stick.



Stickase with a magnet-lined pocket complementary in structure to the stick (the substrate) stabilizes the substrate. Bending is impeded by the magnetic attraction between stick and stickase.



An enzyme with a pocket complementary to the reaction transition state helps to destabilize the stick, contributing to catalysis of the reaction. The binding energy of the magnetic interactions compensates for the increase in free energy required to bend the stick.



The binding energy that provides energy for catalysis also gives an enzyme its specificity.



Specificity of an enzyme: the ability to discriminate between a substrate and a competing molecule.



If an enzyme active site has functional groups arranged such that a variety of weak interactions are optimized with a particular substrate in the transition state, then enzyme will not be able to interact to the same degree with any other molecule.



Specificity is derived from the formation of many weak interactions between the enzyme and its specific substrate molecule.



Following are 4 physical and thermodynamic factors that contribute to the activation energy (ΔG‡) and the mechanisms used by enzymes to counter them.

1. A reduction in entropy, in the form of decreased freedom of motion of two molecules in solution



To counter this enzymes provide the binding energy that holds the substrates in the proper orientation to react.



Substrates can be precisely aligned on the enzyme, with many weak interactions between E and S, clamping the substrate molecules into the proper positions.

2. The solvation shell of hydrogen-bonded water that surrounds and helps to stabilize most biomolecules in aqueous solution

To counter this enzyme forms weak bonds with the substrate. This results in desolvation of the substrate.

3. The distortion of substrates that must occur in many reactions.

binding energy involving weak interactions formed only in the reaction transition state helps to compensate thermodynamically for any distortion, primarily electron redistribution that the substrate must undergo to react.

4. The need for proper alignment of catalytic functional groups on the enzyme.

enzyme itself usually undergoes a change in conformation when the substrate binds. This is called as Induced fit (Koshland).

fit brings specific functional groups on the enzyme into the proper position to catalyze the reaction.

Mechanisms of enzyme catalysis

1. General acid-base catalysis

charged intermediates

intermediates

be stabilized

in a reaction

breakdown to constituent reactants.

transfer

form a species that breaks down more readily

or from the substrate

intermediate

Acid-Base catalysis:

donors.

non-enzymatic

proton transport is from water molecules or weak proton acceptors

General Acid-Base catalysis: proton transfers mediated by other classes of molecules (other than water). Weak organic acids acts as proton donors and weak organic bases act as proton acceptors.

2. Covalent catalysis

A transient covalent bond is formed between the enzyme and the substrate. Consider the following hydrolysis reaction:

In the presence of a covalent catalyst (an enzyme with a nucleophilic group X:) the reaction becomes

This new pathway has lower activation energy as compared to the 1st uncatalyzed pathway. Many amino acid side chains and the functional groups of some enzyme cofactors can serve as nucleophiles.

The transient covalent bond formed between E and S can activate substrate for further reaction. Metal ion catalysis

Ionic interactions are formed between an enzymebound

This helps to orient the substrate for reaction or stabilize the charged reaction transition states.

Metals can also mediate oxidation-reduction reactions by reversible changes in the metal ion’s oxidation state. (Nearly a third of all known enzymes require one or more metal ions for catalytic activity).

Most enzymes use a combination of several catalytic strategies to bring about catalysis. For example, chymotrypsin uses both general acidbase catalysis and covalent catalysis.

The first step in the reaction catalyzed by chymotrypsin is the acylation step. The hydroxyl group of Ser aided by general base catalysis (the base is the side chain of His

Enzyme kinetics











Figure1.6.4.Covalentandgeneralacid-base thereactioncatalyzed by The hydroxyl in areaction generalbasecatalysis(thebase is the ).Thisprovidesanewpathway apeptidebond. in thenew fasterthantheuncatalyzedreaction. Visit:http://study.biotecnika.orgforcoloredpicture.

Enzyme kinetics: A study of the mechanism of an enzyme-catalyzed reaction to determine the rate of the reaction and how it changes in response to changes in experimental parameters.

Substrate concentration [S] is a key factor which determines the rate of the reaction.

In an experimental set up it is difficult to study the effect of substrate concentration on rate of reaction as the substrate depletes with the reaction progress.

A simplified approach is to measure the initial rate/velocity (Vo), when [S] is much greater than the concentration of enzyme, [E].

If only the beginning of the reaction is monitored (often the first 60 seconds or less), changes in [S] can be limited to a few percent, and [S] can be regarded as constant.

The Vo can be plotted as a function of [S] and the following plot is observed. (Figure 1.6.5.)

At low [S], Vo increases linearly with [S].

At high [S], increase in Vo decreases with respect to increase in [S].

Finally at very high [S] concentration, plateau-like V0 region is formed which is close to the maximum velocity, Vmax.

The maximum initial rate of the catalyzed reaction (Vmax) is observed when at very high substrate concentrations, the enzyme is saturated i.e. all E is present in the form of ES and free E is very small.

Pre-steady state:

The initial period when the E is 1st mixed with large concentration of S, during which the concentration of S builds up. (very short period, lasts only a few microseconds)

Steady state: [ES] (and the concentrations of any other intermediates) remains approximately constant over time.

Derivation of Michaelis-Menten Equation: (rate equation for 1 substrate-enzyme catalyzed reaction)

Consider the following reaction,

Where, k

k-1 = rate constant for breakdown of ES

k2 = rate constant for formation of E + P

substrateconcentration enzyme-catalyzed

k-2 can be ignored as in the initial stages of reaction, concentration of P is negligible, so P→S is negligible.

V0 is determined by the breakdown of ES to form product, which is determined by [ES].

[ES] is difficult to measure, so an alternative term has to be found out.

If Et is the total enzyme concentration then,

Concentration of bound E = [ES]

Concentration of unbound E = Et

The rates of formation and breakdown of ES are determined by their respective rate constants.

Steady state Assumption is that at steady state rate of ES formation is equal to the rate of ES breakdown.

The left side is multiplied out and the right side is simplified to give

Adding the term k1[ES][S] to both sides of the equation and simplifying gives

Solving this equation for [ES]:

Further simplifying and combining the rate constants into one expression:

The term (k2 + k-1)/k1 is defined as the Michaelis constant, Km. So the equation becomes,

Expressing V0 in terms of [ES]

substituting [ES] in the equation Vo = k2 [ES],

The maximum velocity occurs when the enzyme is saturated (i.e. when [Et] = [ES] ), Vmax = k2[Et].

this in the above equation we get the Michaelis-Menten equation:

a special case where,

is exactly one half of Vmax. Then the Michaelis-Menten equation

dividing by Vmax

Solving for Km,

Definition of Km

get,

get Km

[S] = 2[S],

Km is equivalent to the substrate concentration at which V

The dependence of initial velocity on substrate concentration can be explained with the graph provided below.

is a simple graphical method for obtaining an approximate value for Km.

Lineweaver- Burk Plot : Burk plot (or double reciprocal plot) is a graphical representation of the Lineweaver

Figure1.6.7.Adouble-reciprocal or Lineweaver-Burkplot.

Interpreting Vmax and Km:

Figure1.6.6.Dependence of initialvelocity on substrateconcentration.

Km can vary greatly from enzyme to enzyme, and even for different substrates of the same enzyme.

It is sometimes used (often inappropriately) as an indicator of the affinity of an enzyme for its substrate.

Actual meaning of Km changes depending upon the experimental conditions.

For reactions with two steps,

When k2 is rate limiting, k2  k-1

Km reduces to k-1 /k1, which is defined as the dissociation constant, Kd , of the ES complex. Where these conditions hold, Km does represent a measure of the affinity of the enzyme. for its substrate in the ES complex. However, this scenario does not apply for most enzymes.

Sometimes k2  k-1 , then Km = k2 /k1

In other cases, when k2 and k-1 are comparable Km is a complex function of all three rate constants k1, k2 and k-1

The quantity Vmax also varies greatly between different enzymes. If an enzyme reacts by two-step mechanism, the Vmax = k2[Et]

If we consider a three step reaction such as,

Then, Vmax = k3[Et]

So, a more general rate constant (kcat) is defined to describe the limiting rate of any enzyme-catalyzed reaction at saturation.

If the reaction has several steps and one is clearly rate limiting, kcat is equivalent to the rate constant for that limiting step.

For example, for a two step reaction, kcat = k2

For a three step reaction kcat = k3, and so on.

The modified Michaelis-Menten equation

The constant kcat is a first-order rate constant and hence has units of reciprocal time. It is also called the turnover number.

Turnover number: It is equivalent to the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with substrate.

Comparing catalytic mechanisms and efficiencies using kcat and Km:

When [S] << Km,

Becomes

The above equation is a second order rate equation.

The term kcat/Km is the Specificity constant.

It is a second-order rate constant with units of M- -1. It is the rate constant for the conversion of E + S to E + P.

Bimolecular Substrate

Enzymatic reactions with two substrates usually involve transfer of an atom or a functional group from one substrate to the other.

reactions proceed by one of several different pathways.

In some cases, both substrates are bound to the enzyme concurrently at some point in the course of the reaction, forming a noncovalent ternary complex.

The substrates bind in a random sequence or in a specific order which is shown in the diagram below.

we perform steady-state kinetic analysis of bisubstrate reactions which involves formation of ternary complex then, the double reciprocal plot obtained for such a reaction is as follows (Intersecting lines indicate that a ternary complex is formed in the reaction):

Figure1.6.8. The enzymeandbothsubstrates cometogether to formaternarycomplex. In orderedbinding,substrate1mustbindbefore substrate2 can bindproductively. In random binding,thesubstrates can bind in eitherorder.

In other cases, the first substrate is converted to product and dissociates before the second substrate binds, so no ternary complex is formed.

 An example of this is the Ping-Pong, or double-displacement, mechanism as depicted in diagram below.



If we perform steady-state kinetic analysis of bisubstrate reactions which does not involve formation of ternary complex then, the double reciprocal plot obtained for such a reaction is as follows (parallel lines indicate a Ping-Pong or double-displacement pathway)

Figure1.6.9. An enzyme-substratecomplexforms,aproduct leavesthecomplex,thealteredenzymeformsasecondcomplex withanothersubstratemolecule,andthesecondproductleaves, regeneratingtheenzyme.Substrate1maytransferafunctional group to theenzyme(toformthecovalentlymodified E’), which is subsequentlytransferred to substrate2.This is calledaPingPong or double-displacementmechanism.

Enzyme inhibition



Enzyme inhibitors are molecular agents that interfere with catalysis, slowing or halting

Figure 1.6.11. Mechanism of competitive inhibition.

Enzymes can be reversibly or irreversibly

Figure1.6.10.Steady-statekineticanalysis of bisubstratereactions. In thesedouble-reciprocal plots,theconcentration of substrate1 is varied whiletheconcentration of substrate2 is held constant.This is repeatedforseveralvalues of [S],generatingseveralseparatelines.(a) Intersectinglinesindicatethataternarycomplex is formed in thereaction;(b)parallellines indicateaPing-Pong(double-displacement) pathway.

There are three types of reversible inhibition

Figure 1.6.12. Kinetics of competitive inhibition.

Competitive Inhibition:

A competitive inhibitor (I) competes with the substrate for the active site of an enzyme. (I) occupies the active site and prevents binding of substrate to enzyme.

Many competitive inhibitors are similar in structure to substrate.

Competitive inhibition can be analyzed quantitatively by steady-state kinetics.

In the presence of a competitive inhibitor, the Michaelis-Menten equation becomes

Because the inhibitor binds reversibly to the enzyme at the same active site as that of substrate, the inhibitor can be removed from competition simply by increasing the concentration of the substrate.

So Vmax for this reaction remains the same. But the [S]at which Vo = half Vmax, the apparent Km, increases in the presence of inhibitor by the factor α.

(Km increases as inhibitor reduces affinity of E to the S). The double-reciprocal plot is as shown in 1.6.12.

Uncompetitive inhibition:

Uncompetitive inhibitor the substrate active site to the ES complex

In the presence of an uncompetitive inhibitor, the Michaelis-Menten equation is altered to

Figure 1.6.13. Mechanism of uncompetitive

At high concentrations of substrate, Vo approaches Vmax/α’.

Thus, an uncompetitive inhibitor lowers the measured Vmax.

Apparent Km also decreases, because the [S] required to reach oneKm decreases because, as I binds only to ES complex, it appears as if it is increasing the affinity between E and S).

The double reciprocal plot is as shown in Figure 1.6.14.

Mixed Inhibition:

Figure 1.6.14. Kinetics of uncompetitive inhibition.

Mixed inhibitor binds at a site distinct from the substrate active site, but it binds to either E or ES.

The rate equation describing mixed inhibition is

A mixed inhibitor usually affects both Km and Vmax.

Vmax decreases by the factor α’. Km can either increase or decrease depending upon whether I binds to E or ES. If I binds only to E, then Km increases.

I binds only to ES then Km decreases. The double reciprocal plot is as follows:

Non-competitive Inhibition:

Non-competitive inhibition is a special case of mixed inhibition where α = α’

The double reciprocal plot is

In the rate equation of mixed inhibition

Figure 1.6.15. Mechanism of mixed inhibition.

If we substitute α = α’, Vmax decreases by a factor of α (or α’), but Km remains the same.

Irreversible inhibition:

Irreversible inhibitors bind covalently with or destroy a functional group on an enzyme that is essential for the enzyme’s activity, or form a particularly stable noncovalent association.

Suicide inactivators:

A Special class of irreversible inhibitors which are relatively un reactive until they bind to the active site of a specific enzyme.

Such a compound gets converted to a very reactive compound when combined irreversibly with the enzyme.

These compounds are also called mechanism-based inactivators, because they hijack the normal enzyme reaction mechanism to inactivate the enzyme.

Dependence of Enzyme activity on pH

Figure 1.6.16. Kinetics of mixed inhibition.

Enzymes have an optimum pH (or pH range) at which their activity is maximal. At higher or lower pH, activity decreases.

Comparing the pH activity profile of 2 enzymes:

Pepsin, which hydrolyzes certain peptide bonds of proteins during digestion in the stomach, has a pH optimum of about 1.6. The pH of gastric juice is between 1 and 2

Glucose 6-phosphatase of hepatocytes (liver cells), with a pH optimum of about 7.8, is responsible for releasing glucose into the blood. The normal pH of the cytosol of hepatocytes is about 7.2.

Regulatory enzymes

In cells, many enzymes work together to carry out a given metabolic process. In such systems, the product of one enzyme becomes the substrate for the next enzyme.

The regulatory enzymes exhibit increased or decreased catalytic activity in response to certain signals.

In most multi-enzyme systems, the first enzyme of the sequence is a regulatory enzyme.

Allosteric enzymes function through reversible, noncovalent binding of regulatory compounds called allosteric modulators or allosteric effectors, which are generally small metabolites or cofactors

Other enzymes are regulated by reversible covalent modification.

Metabolic systems have at least two other mechanisms of enzyme regulation:

1. Some enzymes are stimulated or inhibited when they are bound by separate regulatory proteins.

Figure 1.6.17. Kinetics of Non-competitive Inhibition

2. Some are activated when peptide segments are removed by proteolytic cleavage; unlike effector-mediated regulation, regulation by proteolytic cleavage is irreversible.

Allosteric enzymes undergo conformational changes in response to modulator binding.

Conformational changes induced by one or more modulators interconvert more active and less-active forms of the enzyme.

The modulators for allosteric enzymes may be inhibitory or stimulatory.



Often the modulator is the substrate itself; regulatory enzymes for which substrate and modulator are identical are called homotropic.

When the modulator is a molecule other than the substrate, the enzyme is said to be heterotropic.

Each regulatory site is specific for its modulator. Enzymes with several modulators generally have different specific binding sites for each.

In homotropic enzymes, the active site and regulatory site are the same.

Feedback Inhibition:

In some multienzyme systems, the regulatory enzyme is specifically inhibited by the end product of the pathway whenever the concentration of the end product requirements.

The rate of production of the pathway’s end product is thereby brought into balance with the cell’s needs. Build-up of the end product slows the entire pathway.

In the the conversion of L-threonine to L-isoleucine in bacterial systems, the first enzyme, threonine dehydratase, is inhibited by isoleucine, the product of the last reaction of the series. This is an example of heterotropic allosteric inhibition.

Kinetic properties of Allosteric enzymes:

Kinetic properties of Allosteric enzymes differ from MichaelisMenten kinetics.

Figure1.6.18.Feedback inhibition. The conversion of Lthreonine to L-isoleucine.

Allosteric enzymes do show saturation with substrate but for some allosteric enzymes, plots of Vo versus [S] produce a sigmoid saturation curve, rather than the hyperbolic curve typical of non-regulatory enzymes.

In this plot the value of [S] at Vo=halfVmax is not referred to as Km, instead the term [S]0.5 or K0.5 is used.

Sigmoid kinetic behavior generally reflects cooperative interactions between protein subunits.

For most Homotropic allosteric enzymes, the substrate acts as a positive modulator (an activator) and the binding of one molecule of substrate to one binding site alters the enzyme’s conformation and enhances the binding of subsequent substrate molecules. In sigmoid kinetics, a small change in the concentration of a modulator can be associated with large changes in activity.

For heterotropic allosteric enzymes, an activator may cause the curve to become more nearly hyperbolic, with a decrease in K0.5 but no change in Vmax, resulting in an increased reaction velocity at a fixed substrate concentration.

Other heterotropic allosteric enzymes respond to an activator by an increase in Vmax with little change in K0.5.

A negative modulator (an inhibitor) may produce a more sigmoid substrate-saturation curve, with an increase in K

Covalent Modification of Regulatory enzymes:

Methylation of bacteria

Transmembrane sensor protein in bacteria permits a bacterium to swim toward an attractant (such as a sugar) in solution and away from repellent chemicals. The methylating agent is S-adenosylmethionine (adoMet).

ADP

Diptheria toxin eEF2 (inhibition of protein synthesis)

Cholera toxinprotein (inhibition of signaling pathway)

Phosphorylation - This mode of covalent modification is central to a large number of regulatory pathways.

Protein Kinases: Attach phosphoryl groups to specific amino acid residues of a protein

Phosphatases: remove phosphoryl groups

Regulation by phosphorylation is seen in glycogen phosphorylase (Mr 94,500) of muscle and liver, which catalyzes the following reaction

alteredandK0.5 is nearly

Glycogen phosphorylase occurs in two forms: the more active phosphorylase a and the less active phosphorylase b.

Phosphorylase has two subunits, each with a specific Ser residue that is phosphorylated at its hydroxyl group.

The phosphoryl groups can be hydrolytically removed by a separate enzyme called phosphorylase phosphatase:

In this reaction, phosphorylase a is converted to phosphorylase b by the cleavage of two serine phosphate covalent bonds, one on each subunit of glycogen phosphorylase.

Phosphorylase b can in turn be reactivated transformed back into active phosphorylase a enzyme, phosphorylase kinase, which catalyzes the transfer of phosphoryl groups from ATP to the hydroxyl groups of the two specific Ser residues in phosphorylase b: Breakdown of Glycogen in liver and skeletal muscles is regulated by variation in the ratio of a and b forms of glycogen phosphorylase.

Regulation of enzyme activity by proteolytic cleavage of enzyme precursor:

Inactive precursor called a zymogen is cleaved toform the active enzyme.

Eg. Proteases of the stomach and pancreas: Chymotrypsin and trypsin are initially synthesized as chymotrypsinogen and trypsinogen.

Cleavage exposes the enzyme active site.

of glycogen by covalent

Figure1.6.24.Activation of zymogens by proteolytic cleavage.Activation of Trypsin.

Figure1.6.23.Activation of zymogens by proteolyticcleavage.Activation of Chymotrypsin.

Proteases are inactivated by inhibitor proteins that bind very tightly to the enzyme active site. For e.g., pancreatic trypsin inhibitor (Mr 6,000) binds to and inhibits trypsin.

Critical thinking Questions

1. Supposeamutantenzymebindsasubstrate100-fold as tightly as doesthenativeenzyme. What is theeffect of thismutation thecatalyticrate if thebinding of thetransition state

2. What is athiolprotease?

3. TheHIV1protease,likeotherretroviralproteases, thanasinglechaintwice arrangement?

4. Youhaveisolatedadimericenzymethatcontains2identicalactivesites.Thebinding substrate allostericmodelbestaccountsforthisnegativecooperativity?

5. Antithrombin What

CONFORMATION OF PROTEINS

Amino Acids

Amino acids are the building blocks of protein.

The primary functions of amino acids are to build muscle tissue.

More than 300 amino acids exist in nature.

Only 20 aa are used by the living organisms.

10 Amino acids can be synthesized by the body itself.

All 20 of the common amino acids are amino acids.

Structure of Amino acid

Amino acids differ from each other in their side chains/R groups

electric charge, and which influence the solubility of the amino acids in water. All the common amino acids except glycine, the carbon is bonded to four different groups:

1. a carboxyl group

2. an amino group

3. an R group

4. a hydrogen atom

The -carbon atom is thus a chiral center. So they are optically active. In glycine, the R group is another hydrogen, so optically inactive They form enantiomers. The Amino Acid residues in proteins are L-stereoisomers

Classification of Amino Acids

They are classified based on the properties of their R groups

Polar amino acids

Figure1.7.1Generalstructure of an aminoacid.Thisstructure is common to all butoneof the-aminoacids.(Proline, acyclicaminoacid, is theexception.)

TheRgroup or sidechainattached to the

α carbon is different in eachaminoacid.

-aminoacids.

Non-polar amino acids Glycine, Valine, Alanine, proline, Leucine, Isoleucine, Methionine

Amino acids with negatively charged R group

Amino acids with Positively charged R group-

Amino acids with Aromatic R group-

Classification of Amino Acids -Based on functional group

charged

charged

Aliphatic: gly (G), ala (A) , val (V), leu (L), ile (I)

Aromatic: Trp (W), Phe (F), Tyr (Y), His (H),

Polar

1.7.3

ionization

20 common amino acids

proteins.

structural formulas show the state

Uncommon Amino Acids

plant

constituent

wall

bloodclotting protein

a derivative of four Lys - Elastin.

2+ binding proteins

introduced during protein synthesis rather than created through a postsynthetic

contains selenium

than the sulfur of cys. It is present at the catalytic site of

glutathione

dehydrogenase

of Arginine and urea cycle.

Amino Acids Can Act as Acids and Bases

When an amino acid is dissolved in water, it exists in solution as the dipolar ion, or zwitterion.

A zwitterion can act as either an acid (proton donor) or a base (proton acceptor)

Substances having this dual nature are are often called ampholytes

pI of Amino Acids

Amphoteric molecules called zwitterions. The net charge on the molecule is affected by pH of their surrounding environment and can become more positively or negatively charged due to the loss or gain of protons (H

The pI is the pH value at which the molecule carries no electrical charge or the negative and positive charges are equal.

a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge.

Amino acids are ampholytes, pKa of α is =2 and of α

The compared to NH3 group.

Peptide bond Formation

Two amino acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond, to yield a dipeptide.

It is formed by removal of water (dehydration) from the α carboxyl group of one amino acid and the α amino group of another.

It is a type of condensation reaction.

It is an Endergonic reaction with ΔG=+21KJ/mol.

ofan aminoacid.Shownhere is thetitrationcurve of 0.1Mglycine at25 C.The ionicspeciespredominating at keypoints in the titrationareshownabovethegraph.

Properties of Peptide Bond

Peptide Bond Angles

than the C N bond in a simple amine and that the atoms associated with the peptide bond

This indicated a resonance or partial sharing of two pairs of electrons between the carbonyl oxygen and the amide nitrogen.

of apeptidebond by condensation.

Why Partial Double bond?

The oxygen has a partial negative charge and the nitrogen a partial positive charge, setting up a small electric dipole.

6 atoms of the peptide group lie in a single plane, with the oxygen atom of the carbonyl group and the hydrogen atom of the amide nitrogen trans to each other.

Bond Denotation

Proteins

Protein Conformation

The spatial arrangement of atoms in a protein is called its conformation. The possible conformations of a protein include any structural state that can be achieved without breaking covalent bonds. A change in conformation could occur by rotation about single bonds. Of the numerous conformations that are theoretically possible in a protein containing hundreds of single bonds, a few generally predominate under biological conditions. The need for multiple stable conformations reflects the changes that must occur in most proteins as they bind to other molecules or catalyze reactions. The conformations existing under a given set of conditions are usually the ones that are thermodynamically the most stable, having the lowest Gibbs free energy (G).

Proteins in any of their functional, folded conformations are called

A protein’s conformation is stabilized largely by weak interactions

Ramachandran Plot

plotted in a Ramachandran plot

values for

introduced by G. N. Ramachandran

Bond Angles For Different Proteins Structure of Proteins

Primary structure

Secondary structure

Tertiary structure

structure

Structures

sequence

regular chain organization pattern

complex folding

The helix is stabilized by H-bonds between N-H and C=O groups of every 4th amino acid

amino acid residues in the helix have conformations with

= 45 to 50 and

= 60

Role of Amino Acids in Alpha Helix formation

Five different kinds of constraints affect the stability of an

helix

the electrostatic repulsion (or attraction) between successive

Figure1.7. Anti-parallel Sheets;TheParallelBeta-Sheet is characterized by twopeptidestrandsrunning in thesamedirectionheldtogether by hydrogenbondingbetween the strands;TheAntiparallelBeta-Sheet is characterized by twopeptidestrandsrunning in oppositedirectionsheldtogether by hydrogen bondingbetween the strands.

ccurrence of Pro and Gly residues

the interaction between amino acid residues at the ends of the helical segment and the electric dipole inherent to the helix

long block of Glu residues cannot form

helix negatively charged carboxyl groups of adjacent Glu residues repel each other

long block of Lys and/or Arg residues- cannot form

helix positively charged carboxyl groups of adjacent residues repel each other

The bulk and shape of Asn, Ser, Thr, and Cys - can also destabilize an α helix if they are close together in the chain.

Ion pair- Positively charged amino acids are often found three residues away from negatively charged amino acids, permitting the formation of an

Two aromatic amino acid residues are often similarly spaced, resulting in a hydrophobic interaction

Pro residue introduces a destabilizing kink in an α helix

Glycine occurs infrequently in α helices as it has more conformational flexibility than the other amino acid residues.

The four amino acid residues at each end of the helix do not participate fully in the helix hydrogen bonds

The partial positive and negative charges of the helix dipole actually reside on the peptide amino and carbonyl groups near the amino-terminal and carboxyl-terminal ends of the helix, respectively.

Negatively charged amino acids are often found near the amino terminus of the helical segment, where they have a stabilizing interaction with the positive charge of the helix dipole; a positively charged amino acid at the amino terminal end is destabilizing.

β-Pleated Sheet

Extended stretches of 5 or more aa are called β

β-strands orga

If adjacent strands are oriented in the same direction (N-end to C-end), it is a parallel β-sheet, if adjacent strands run opposite to each other, it is an antiparallel β sheet. There can also be mixed β sheets

H-bond

β-sheets are usually twisted rather than flat

acid binding proteins are made almost entirely of β

Bend / Loop

chains can fold upon themselves forming a bend or a loop.

Usually 4 aa are required to form the turn

H-bond between the 1st and 4th aa in the turn

Bends are usually on the surface of globular proteins

Proline residues frequently found in bends / loops

Tertiary Structure

3D folding or ‘bundling up’ of the protein.

residues are buried inside, polar residues are exposed outwards to aqueous environment

formed by thepacking of proteinsecondarystructureelementsintocompactglobularunitscalledproteindomains.

Many proteins are organized into multiple ‘domains’

Domains are compact globular units that are connected by a flexible segment of the polypeptide

Each domain is contributes a specific function to the overall protein

Different proteins may share similar domain structures, eg: kinase-, cysteine-rich-, globin-domains.

5 kinds of bonds stabilize tertiary structure

1. H-bonds

2. van der waals interactions

3. hydrophobic interactions

ionic interactions

5. disulphide linkages

In disulphide linkages, the SH groups of two neighboring cysteines form a S-S- bond called as a disulphide linkage. It is a covalent bond, but readily cleaved by reducing agents that supply the protons to form the SH groups again. Reducing ag helices and β sheets generally are found in different structural layers. This is because the backbone of a polypeptide segment in the β

Domains

Polypeptides with more than a few hundred amino acid residues often fold into two or more stable, globular units called

a domain retains its correct three-dimensional structure even when it is separated (for example, by proteolytic cleavage) from the remainder of the polypeptide chain.

A protein with multiple domains may appear to have a distinct globular lobe for each domain, but, more commonly, extensive contacts between domains make individual domains hard to discern.

Different domains often have distinct functions, such as the binding of small molecules or interaction with other proteins. Small proteins usually have only one domain (the domain is the protein).

Supersecondary Structure/ Motifs/ Folds

Are stable arrangements of several elements of secondary structure and the connections between them

range from simple to complex, sometimes appearing in repeating units or combinations

A single large motif may comprise the entire protein e.g. the coiled coil of α-keratin

formed to bury hydrophobic amino acid R groups so as to exclude water [requires at least two layers of secondary structure].

Classification

Simple

types-

Quaternary Structure

Association

protein

than

homopolymeric

Structure Prediction

heteropolymeric.

Individual aa have a preference for specific 2D structure

No definite rules for 3D structure. Determined by overall sequence and tertiary interactions between remote residues; decrease in free energy.

based on computer calculations and comparison to similar domains of known structure

Post Translational Modifications

During synthesis proteins can incorporate only each of the 20 aa

Many amino acids can be enzymatically modified after incorporation into proteins

phosphorylation of S, T, Y serve as regulatory switches

Amino-terminal acetylation prevents degradation

Glycosylation and fatty acylation makes proteins respectively more hydrophilic or hydrophobic

Protein stability is enhanced by hydroxylation of P in collagen and carboxylation of E in prothrombin

Functional Proteins

1. Collagen

repeat of Tropocollagen

Triple helix structure

handed alpha chains

amino acids

unit- tropocollagen

alpha chains

in right handed

repeats of tripeptide

hydroxylysine(

Crosslinking in Collagen

cross links

Reactive

of allysine

chains of aldehydes

hydroxylase)

amino

lysine by lysyl oxidase

dependant enzyme)

linked

Intermolecular cross linking of tropocollagen

Synthesis of collagen

and entry of polypeptide into lumen of rough ER

Hydroxylation of prolyl and lysyl residues

Glycosylation

of triple helix procollagen

into transport vescicle- Exocytosis

Cleavage & formation of tropocollagen

Lateral covalent cross linking of tropocollagen

of fibrils

2.Elastin

Highly hydrophobic connective tissue.

Extensibility and elasticity

72KDa tropoelastin

Rich in nonpolar amino acids

High proline and glycine content

No glycosylation

hydroxyproline but no hydroxylysine

residues of tropoelastin

oxidase -oxidative deamination

aldehydes+1 lysine

formation

4.Alpha-Keratin

Figure 1.7.14 Structure of Desmosine

almost the entire dry weight of hair, wool, nails, claws, quills, horns, hooves, and much of the outer layer of skin

Alpha-keratin is rich in the hydrophobic residues Ala,Val, Leu, Ile, Met, and Phe.

The -keratin helix is a right-handed

strands of -keratin, oriented in parallel (with their amino termini at the same end), are wrapped about each other to form a supertwisted coiled coil.

5.Intermediate filament of eukaryotes

Alpha keratin- mammals

Bete keratin- birds and reptiles

keratin- hetero dimer of TypeI (acidic) and Type II (neutral or basic)

coil is left handed

heterodimers antiparallely join to form a tetramer (protofilament)

protofilaments- protofibril

protofibrils- microfibril

microfibrils macrofibril

in cysteine

has been dealt in Respiratory System of Unit 7

Critical thinking Questions

1. How can curls be induced in hair?

2. Supposethataprotease is synthesized by thesolid-phasemethodfromDratherthanL aminoacids.Howwouldthesedimentation,electrophoreticandcirculardichroism properties of thisenzymecomparewiththose of thenativeform?

3. Proteinsthatspanbiologicalmembranesoftencontainalphahelices.Why is an alpha-helix particularlysuited to exist in ahydrophobicenvironment?

4. What is theessentialconditionforaprotein to be identical to anotherprotein?

5. In sicklecellanemia,areall of thestructurallevels of theproteinmodified?

Wehave learnt in previous section about the Nucleic acids and their structure. Deoxyribonucleic acid (DNA) contains all the information required to build the cells and tissues of an organism. The exact replication of this information in any species assures its genetic continuity from generation to generation and is critical to the normal development of an individual. The information stored in DNA is arranged in hereditary units, now known as genes that control identifiable traits of an organism. In the process of transcription, the information stored in DNA is copied into ribonucleic acid (RNA), which has three distinct roles in protein synthesis.

Discovery of the structure of DNA in 1953 and subsequent elucidation of how DNA directs synthesis of RNA, which then directs assembly of proteins dogma were monumental achievements marking the early days of molecular biology. However, the simplified representation of the central dogma as DNA protein does not reflect the role of proteins in the synthesis of nucleic acids. Moreover, as discussed in later chapters, proteins are largely responsible for regulating gene expression, the entire process whereby the information encoded in DNA is decoded into the proteins that characterize various cell types.

We have seen in detail the structure of nucleic acids, and hence we shall directly poop into the further details of its polymerization.

Figure1.8.1Thechemicalstructureshowsahydroxylgroup

3

the 5’ end.Note also

referred to as a phosphodiester

By convention,apolynucleotide

alwayswritten in the 5n3direction(left to right)

Native DNA Is a Double Helix of Complementary Antiparallel Strands. The modern era of molecular biology began in 1953 when James D. Watson and Francis H. C. Crick proposed that DNA has a double-helical

structure. Their proposal, based on analysis of x-ray diffraction patterns coupled with careful model building, proved correct and paved the way for our modern understanding of how DNA functions as the genetic material.

Chargaff’s Rule:

Two Strands have complementary sequences

2 logical operations to obtain complementary strand 5' to 3'

1.Reverse: Rewrite the sequence, back to front

2.Complement: Swap A with T, C with G

1.8.1 B DNA

DNA consists of two associated polynucleotide strands that wind together to form a double helix. The two sugar phosphate backbones are on the outside of the double helix, and the bases project into the interior. The adjoining bases in each strand stack on top of one another in parallel planes. The orientation of the two strands is antiparallel; that is, their 5’ 3’ directions are opposite. The held in precise register by formation of base pairs between the two strands: A is paired with T through two hydrogen bonds; G is paired with C through three hydrogen bonds. This base-pair complementarity is a consequence of the size, shape, and chemical composition of the bases. The presence of thousands of such hydrogen bonds in a DNA molecule contributes greatly to the stability of the double helix.



Hydrophobic and van der Waals interactions between the stacked adjacent base pairs further stabilize the double-helical structure.



In natural DNA, A always hydrogen bonds with T and G with C, forming A·T and G·C base pairs as shown in figure.

Chargaff’s

rule:AlwaysApairswith T/UandGpairswithC. Complementarity of theDNA

Thecomplementary antiparallelstrands of DNAfollowthepairing rulesproposed by WatsonandCrick.The base-pairedantiparallel in base composition:theleft strandhas the compositionA3T2G1 right,A2T3G3 C1.Theyalsodiffer in sequencewheneach in the 5n3 the base equivalences:ATandG duplex.

 These associations between a larger purine and smaller pyrimidine are often called Watson-Crick base pairs



Two polynucleotide strands, or regions thereof, in which all the nucleotides form such base pairs are said to be complementary.



However, in theory and in synthetic DNAs other base pairs can form. For example, a guanine (a purine) could theoretically form hydrogen bonds with a thymine (a pyrimidine) causing only a minor distortion in the helix.



The space available in the helix also would allow pairing between the two pyrimidines cytosine and thymine. Although the nonstandard G·T and C·T base pairs are normally not found in DNA, G·U base pairs are quite common in double-helical regions that form within otherwise single-stranded RNA

The Most DNA in cells is a right-handed helix. The stacked bases are regularly spaced 0.36 nm apart along the helix axis. helix makes a complete turn every 3.6 nm; thus there are about 10.5 pairs per turn.

 This is referred to as the B form of DNA, the normal form present in most DNA stretches in cells.

On the outside of B-form DNA, the spaces between the intertwined strands form two helical grooves of different widths described as the major groove and the minor groove

As a consequence, the atoms on the edges of each base within these grooves are accessible from outside the helix, forming two types of binding surfaces. DNA binding Proteins can “read” the sequence of bases in duplex DNA by contacting atoms in either the major or the minor grooves.

Structural variation in DNA reflects three things: the different possible conformations of the deoxyribose, rotation about the contiguous bonds that make up the phosphodeoxyribose backbone (Fig. 1.94 ), and free rotation about the C-1’ N-glycosyl bond (Fig.1.95). Because of steric constraints, purines in purine nucleotides are restricted to two stable conformations with respect to deoxyribose, called syn and anti (Fig. 1.5). Pyrimidines are generally restricted to the anti-conformation because of steric interference between the sugar and the carbonyl oxygen at Cof the pyrimidine

In addition to the major B form, three additional DNA structures have been described. Two of these are compared to B DNA in Figure. In very low humidity, the crystallographic structure of B DNA changes to the A form; RNA-DNA and RNARNA helices exist in this form in cells and in vitro.

1.8.2 Z DNA:



DNA.Theoriginal Å(3.4 nm), .5basepairs,

Short DNA molecules composed of alternating purine-pyrimidine nucleotides (especially G s and Cs) adopt an alternative left-handed configuration instead of the normal right-handed helix. the bases seem to zigzag when viewed from the side.

Some evidence suggests that Z DNA may occur in cells, although its function is unknown.

Z-DNA is quite different from the right-handed forms. In fact, Z-DNA is often compared against B-DNA in order to illustrate the major differences.

The Z-DNA helix is left-handed and has a structure that repeats every 2 base pairs. The major and minor grooves, unlike A- and B-DNA, show little difference in width.

Structural variation in DNA. (a) The conformation of a

DNA is affected by rotation about seven different bonds. Six of the bonds rotate freely. The limited rotation about bond 4 gives rise to ring pucker, in which one of the atoms in the five-membered furanosering is out of the plane described by the other four. This conformation is endoor exo, depending on whether the atom is displaced to the same side of the plane as C-5 or to the opposite side

Formation of this structure is generally unfavourable, although certain conditions can promote it; such as alternating purine-

backboneand

pyrimidine sequence (especially poly(dGC)2), negative DNA supercoiling or high salt and some cations (all at physiological temperature, 37°C, and pH 7.3-7.4).

Z-DNA can form a junction with B-DNA (called a "B-to Z junction box") in a structure which involves the extrusion of a base pair.

purinebases in theattachedribose syn.Pyrimidinesgenerallyoccur in the

The Zconformation has been difficult to study because it does not exist as a stablefeature of the double helix. Instead, it is a transient structure that is occasionally induced by biological activity and then quickly disappears.

Biological significance

It is believed to provide torsional strain relief (supercoiling) while DNA transcription occurs. The potential to form a Z-DNA structure also correlates with regions of active transcription

Toxic effect of ethidium bromide on trypanosomas is caused by shift of their kinetoplastid DNA to Z-form. The shift is caused by intercalation of EtBr and subsequent loosening of DNA structure that leads to unwinding of DNA, shift to Z-form and inhibition of DNA replication.

Z-DNA formed after transcription initiation

Z-DNA is necessary for transcription and prolonging expression of the anti-apoptotic genes.

A DNA

A-DNA is one of the many possible double helical structures of DNA. A-DNA is thought to be one of three biologically active double helical structures along with B-DNA and Z-DNA.

It is a right-handed double helix fairly similar to the more common and well-known B-DNA form, but with a shorter more compact helical structure.

occurs only in dehydrated samples of DNA, such as those used in crystallographic experiments, and possibly is also assumed by DNA-RNA hybrid helices and by regions of double-stranded RNA

Most RNA and RNA-DNA duplex in this form

Shorter, wider helix than B.

Deep, narrow major groove not easily accessible to proteins

Wide, shallow minor groove accessible to proteins, but lower information content than major groove.

Favored conformation at low water concentrations

Base pairs tilted to helix axis and displaced from axis

Sugar pucker C3'-endo (in RNA 2'-OH inhibits C2'-endo conformation)

A number of other sequencestructural variations have been detected within larger chromosomes that may affect the function and metabolism of the DNA segments in their immediate vicinity. For example, bends occur in the DNA helix wherever four or more adenosine residues appear sequentially in one strand. Six adenosines in a row produce a bend of about 18

The bending observed with this and other sequences may be important in the binding of some proteins to DNA.

Palindrome sequences is a word, phrase, or sentence that is spelled identically read either forward or backward; two examples are ROTATOR and NURSES RUN. The term is applied to regions of DNA with inverted repeats of base sequence having twofold symmetry over two strands of DNA. Such sequences are self-complementary within each strand and therefore have the potential to form hairpin or cruciform (cross-shaped) structures. Mirror repeat: The inverted repeat occurs within each individual strand of the DNA. Mirror repeats do not have complementary sequences within the same strand and cannot form hairpin or cruciform structures. Sequences of these types are found.

Figure1.8.7Palindromesandmirrorrepeats.Palindromesare double-strandednucleicacidswithtwofold repeat(shadedsequence) horizontal axis then coloredarrows.A otherhand,hasasymmetricsequencewithin otherrequiresonly

Figure1.8.8Hairpinsandcruciforms.PalindromicDNA (or RNA)sequences can formalternativestructureswith intrastrandbasepairing.WhenonlyasingleDNA(or RNA)strand is involved,thestructure is calledahairpin.



In every large DNA molecule and can encompass a few base pairs or thousands. The extent to which palindromes occur as cruciforms in cells is not known, although some cruciform structures have been demonstrated in vivo in E.coli. Selfcomplementary sequences cause isolated single strands of DNA (or RNA) in solution to fold into complex structures containing multiple hairpins.

Several unusual DNA structures involve three or even four DNA strands. For example,a cytidine residue (if protonated) can pair with the guanosine residue of a GqC nucleotide pair, and a thymidine can pair with the adenosine of an AUT pair (Fig.1.8.7). The N-7, O6, and N6 of purines, the atoms that participate in the hydrogen bonding of triplex DNA, are often referred to as Hoogsteen positions, and the non-Watson-Crick pairing is called Hoogsteen pairing, after Karst Hoogsteen, who in 1963 first recognized the potential for these unusual pairings. Hoogsteen pairing allows the formation of triplex DNAs. The triplexesare most stable at low pH altered from its normal value pKa of 4.2. The triplexes also form most readily within long sequences containing only pyrimidines or only purines in a given strand. Some triplex DNAs contain two pyrimidine strands and one purine strand; others contain two purine strands and one pyrimidine strand.



Four DNA strands can also pair to form a tetraplex (quadruplex), but this occurs readily only for DNA sequences with a very high proportion of guanosine residues (Fig.1.8.10 ). The guanosine tetraplex, or G tetraplex, is quite stable over a wide range of conditions.



A particularly exotic DNA structure, known as H-DNA, is found in polypyrimidine or polypurine tracts that also incorporate a mirror repeat.

A simple example is a long stretch of alternating T and C residues

The H-DNA structure features the triplestranded

In the DNA of living cells, sites recognized by many sequence-specific DNAbinding proteins are arranged as palindromes, and polypyrimidine or polypurine sequences that can form triple helices or even H-DNA are found within regions involved in the regulation of expression of some eukaryotic genes.

In principle, synthetic DNA strands designed to pair with these sequences to form

Figure1.8.10 DNAstructurescontainingthree or fourDNAstrands.Basepairingpatterns in one well-characterizedform of triplexDNA. Basepairingpattern in theguanosinetetraplexstructure.

triplex DNA could disrupt gene expression. This approach to controlling cellular metabolism is of growing commercial interest for its potential application in medicine and agriculture.

1.8.4 tRNA

Transfer ribonucleic acid (tRNA) is a type of RNA molecule that helps decode a messenger RNA (mRNA) sequence into a protein. tRNAs function at specific sites in the ribosome during translation, which is a process that synthesizes a protein from an mRNA molecule. Proteins are built from smaller units called amino acids, which are specified by three-nucleotide mRNA sequences called codons. Each codon represents a particular amino acid, and each codon is recognized by a specific tRNA. The tRNA molecule has a distinctive folded structure with three hairpin loops that form the shape of a three-leafed clover. One of these hairpin loops contains a sequence called the anticodon, which can recognize and decode an mRNA codon. Each tRNA has its corresponding amino acid attached to its end. When a tRNA recognizes and binds to its corresponding codon in the ribosome, the tRNA transfers the appropriate amino acid to the end of the growing amino acid chain. Then the tRNAs and ribosome continue to decode the mRNA molecule until the entire sequence is translated into a protein.



tRNA itself is an RNA molecule with a conserved inverted L structure.



One end of the tRNA contains an anticodon loop which pairs with a mRNA specifying a certain amino acid. The other end of the tRNA has the amino acid attached to the 3' OH group via an ester linkage.

 tRNA with an attached amino acid is said to be "charged". The enzyme that attaches the amino acid to the 3'-OH is called an aminoacyl tRNA synthetase (aaRS).



There is a specific tRNA for each amino acid, 20 in all. Similarly, there is a specific aaRS for each tRNA.



Only the first 2 nucleotides in the tRNA anticodon loop are strictly required for the decoding of the mRNA codon into an amino acid. The third nucleotide in the anticodon is less stringent in its base-pairing to the codon, and is referred to as the "wobble" base.



Since the genetic code is degenerate, meaning that more than one codon can specify a single amino acid, the anticodon of tRNA can pair with more than one mRNA codon and still be specific for a single amino acid.



To understand how tRNAs can serve as adaptors in translating the language of nucleic acids into the language of proteins, we must first examine their structure in more detail.



Transfer RNAs are relatively small and consist of a single strand of RNA folded into a precise three-dimensional structure. The tRNAs in bacteria and in the cytosol of eukaryotes have between 73 and 93 nucleotide residues, corresponding to molecular weights of 24,000 to 31,000.

of yeast in 1965 by itis shown in which intrastrandbasepairing is maximal.Thefollowing symbolsareused forthe modifiednucleotides:, pseudouridine; I, inosine; T, ribothymidine;D,5,6dihydrouridine;m1I, 1-methylinosine;m1G, 1methylguanosine;m2G, N2-dimethylguanosine Theanticodon can recognizethreecodons for alanine(GCA,GCU,andGCC).Note the presence of twoGUUbasepairs,signified by ablue dot to indicate non-Watson-Crickpairing. In RNAs, guanosine is oftenbasepairedwithuridine, although the GUUpair is not as stable as the Watson-CrickGmCpair

Figure1.8. structure backbonerepresentnucleotideresidues; linesrepresentbasepairs.TransferRNAsvary in lengthfrom nucleotidesoccur arm. At the anticodonloop,whichalwayscontainsseven unpairednucleotides.TheDarmcontainstwo or threeD(5,6-dihydrouridine)residues, depending armhasonlythreehydrogen-bondedbase pairs. In addition Figure1.8. pyrimidinenucleotide;G*,guanylate methylguanylate

Figure 1.8.13 Three-dimensional structure of yeast tRNAPhededuced from x-ray diffraction analysis.

Mitochondria and chloroplasts contain distinctive, somewhat smaller tRNAs. Cells have at least one kind of tRNA for each amino acid; at least 32 tRNAs are required to recognize all the amino acid codons (some recognize more than one codon), but some cells use more than 32. Yeast alanine tRNA (tRNAAla), the first nucleic acid to be completely sequenced (Fig.1.8.11), contains 76 nucleotide residues, 10 of which have modified bases. Comparisons of tRNAs from various species have revealed many common denominators of structure

Eight or more of the nucleotide residues have modified bases and sugars, many of which are methylated derivatives of the principal bases. Most tRNAs have a guanylate (pG) residue at the 5’ end, and all have the

When drawn in two dimensions, the hydrogen-bonding pattern of all tRNAs forms a cloverleaf structure with four arms; the longer tRNAs have a short fifth arm, or extra arm (Fig. 1.8.12). In three dimensions, a tRNA has the form of a twisted L (Fig.1.8.13 ).

1.8.5 Micro RNA

Small ribonucleic acid (RNA) can act as a specific regulator of gene expression. This discovery has been an exciting breakthrough in Biological Sciences of the past decade, culminating in last year’s Nobel Prize in Physiology or Medicine awarded to Andrew Fire and Craig Mello. They discovered that exogenous doublestranded RNA can be used to specifically interfere with gene function. This phenomenon was called RNA interference (RNAi). They also speculated that organisms might use double-stranded RNA naturally as a way of silencing genes. It was then shown that RNA interference was mediated by 22 nucleotide single-stranded RNAs termed small interfering RNAs (siRNAs) derived from the longer double-stranded RNA precursors. The small interfering RNAs were found to repress genes by eliminating the corresponding messenger RNA transcripts, and thus, preventing protein synthesis.



Several hundred genes in our genome encode small functional RNA molecules collectively called microRNAs (miRNAs).

 Precursors of these miRNA molecules form structures of double-stranded RNA that can activate the RNA interference machinery.

MicroRNAs down regulate gene expression either by degradation of messenger RNA through the RNA interference pathway or by inhibiting protein translation.

The first miRNA was discovered in 1993 by Victor Ambros and colleagues Rosalind Lee and Rhonda Feinbaum

A genetic screen in the roundworm Caenorhabditis elegans, a millimetre-long animal used as a model organism in biological research, identified genes involved in developmental timing

Surprisingly, one of the genes, termed lin-4, did not encode a protein but instead a novel 22nucleotide small RNA. Seven years later, it was discovered that a second 22-nucleotide small RNA of this type, let-7, a gene also involved in elegans

The lin-4 and let-7 small regulatory RNAs soon became very exciting for two reasons. Firstly, homologs of the letgene were identified in other animals including humans

The conservation of letacross species suggested an important and fundamental biological role for this small RNA. Secondly, the mechanism of RNA interference (RNAi) was discovered at that time, and it became clear that miRNA and RNAi pathways were intricately linked and shared common components.

These small non-coding RNAs were named microRNAs (miRNAs)

Subsequently, many more short regulatory RNAs were identified in almost all multicellular organisms, including flowering plants, worms, flies, fish, frogs, mammals

To date, more than 500 human miRNAs have been experimentally identified.

miRNAs.aPrimary miRNAs(pri-miRNA)aretranscribedfromlongerencodingDNA oneor more thenucleus,the stem-loopstructure to precursormiRNA(pre-miRNA).bAfterexportinto the theribonucleaseDicer to .cThemature RNA-induced silencingcomplex(RISC),whilethecomplementarystrand (miRNA*) is usuallyrapidlydegraded.ThemiRNAincorporated into the silencingcomplex can bind tothe targetmessengerRNA by basepairing,causinginhibition of proteintranslationand/or degradation of the targetmessenger RNA

This makes miRNAs one of the most abundant classes of regulatory genes in humans. MicroRNAs are now perceived as a key layer of post-transcriptional control within the networks of gene regulation.

MicroRNAs are sequentially processed from longer precursor molecules that are encoded by the miRNA (Fig. 1.8.14).

MiRNA genes are referred to by the same name (termed mir) written in italics to distinguish them from the corresponding mature miRNA (termed miR) followed by a number, e.g., mir-1 or miR-1. The encoding DNA sequence is much longer than the mature miRNA. Two ribonuclease enzymes, Drosha and Dicer, subsequently process the primary transcripts (or pri-miRNA) to generate mature miRNAs.

The primary transcripts contain one or more stem-loop structures of about 70 bases. Stem-loops are double-stranded RNA structures consisting of a nucleotide sequence that can fold back on itself to form a double helix with a region of imperfect base pairing that forms an open loop at the end

The ribonuclease Drosha excises the stem-loop structure to form the precursor miRNA (or pre-miRNA) After export into the cytoplasm, the pre-miRNA is cleaved by the ribonuclease Dicer to generate a short RNA duplex. After untwisting, one RNA strand becomes the mature singlestranded miRNA, while the complementary strand, termed miRNA, is usually rapidly degraded

MicroRNAs recognize their targets based on sequence complementarity. The mature miRNA is partially complementary to one or more messenger RNAs. In humans, the complementary sites are usually within the 3′ the target messenger RNA.

To become effective, the mature miRNA forms a complex with proteins, termed the RNA miRNA incorporated into the silencing complex can bind to the target messenger RNA by base pairing.

This base pairing subsequently causes inhibition of protein translation and/or degradation of the messenger RNA (Fig. 1.8.14c).

Protein levels of the target gene are consequently reduced, whereas messenger RNA levels may or may not be decreased. In humans, miRNAs mainly inhibit protein translation of their target genes and only infrequently cause degradation or cleavage of the messenger RNA

The biological role and in vivo functions of most mammalian miRNAs are still poorly understood. In invertebrates, miRNAs regulate developmental timing (e.g., lin-4), neuronal differentiation, cell proliferation, growth control, and programmed cell death

In mammals, miRNAs have been found to play a role in embryogenesis and stem cell maintenance , hematopoietic cell differentiation and brain

miRNAtranslational repressionandRNAinterferencemediated by the RNA-inducedinterferingcomplex(RISC).Processing bothmiRNAprecursorsintomiRNAsandlong double-strandedRNAsintoshortinterferingRNAs Dicerribonuclease. In both Diceryieldsadouble-stranded nucleotidesper ed tails.

thisintermediateassembleswith RNAinducedsilencing targetmRNA RISCRNA,leading to miRNAfunction,theRISC targetmRNAthat this case blocked.This is the C.elegans. [AdaptedfromG.HutvagnerandP.D.Zamore, 2002, 2001,Cell 107:823.]

To date, knowledge of human miRNAs has been primarily descriptive. MicroRNA expression has been found to be deregulated in a wide range of human diseases including cancer. However, it remains uncertain whether altered miRNA expression is a cause or consequence of pathological processes. The underlying mechanisms of why and how miRNAs become deregulated are largely unknown. Although bioinformatics approaches can predict thousands of genes that are potentially targeted and regulated by miRNAs based on sequence complementarity, only very few miRNA target genes have been functionally validated.

miRNA and siRNA can be used to treat inherited genetic disorders, cancer, obesity …etc.

Critical thinking Questions

1. How does cordycepin (3’-deoxyadenosine) block the synthesis of RNA?

2. A negatively supercoiledDNA molecule undergoes a B to Z transition over a segment of 360 base pairs. What is the effect on the writhing (supercoiling)?

3. Why is HAP column used to distinguish single stranded and doublestranded DNA?

4. To precipitate DNA, an alcohol like ethanol or propanolis added to an aqueous DNA solution. Why should Na+ or NH4+ also be added?

5. A 41.5 nm-long duplex DNA molecule in the B-conformation adopts the A conformation upon dehydration How long is it now? What is its approximate number of base pairs?

1.9 STABILITY OF PROTEINS AND NUCLEIC ACIDS

The three-dimensional structure and the function of proteins can be destroyed by denaturation, demonstrating a relationship between structure and function.

Some denatured proteins can renature spontaneously to form biologically active protein, showing that protein tertiary structure is determined by amino acid sequence.

Protein folding in cells probably involves multiple pathways. Initially, regions of secondary structure may form, followed by folding into super secondary structures. Large ensembles of folding intermediates are rapidly brought to a single native conformation.

For many proteins, folding is facilitated by Hsp70 chaperones and by chaperonins.

Disulfide bond formation and the cis-trans isomerization of Pro peptide bonds are catalysed by specific enzymes.

acid stability depends on Temperature, pH and salt concentrations

nucleic acids show hyper and hypochromacity

on their double or single stranded nature

1.9.1 Stability of Proteins

Protein are polymers of amino acids, as we learnt in last section. All proteins begin their existence on a ribosome as a linear sequence of amino acid residues. This polypeptide must fold during and following synthesis to take up its native conformation. We have seen that a native protein conformation is only marginally stable. Modest changes in the protein’s environment can bring about structural changes that can affect function. We now explore the transition that occurs between the folded and unfolded states.

dichroism,atechniquethat measures the amount of helicalstructure in a macromolecule.Denaturation of ribonucleaseAwastracked by monitoringchanges in the intrinsicfluorescence of the protein,which is affected by changes in the environment of Trpresidues. (b) Denaturation of disulfide-intactribonuclease A by guanidinehydrochloride (GdnHCl), monitored by circular dichroism

Most proteins can be denatured by heat, which affects the weak interactions in a protein (primarily hydrogen bonds) in a complex manner. If the temperature is increased slowly, a protein’s conformation generally remains intact until an abrupt loss of structure (and function) occurs over a narrow temperature range (Figure 1.8.1). The abruptness of the change suggests that unfolding is a cooperative process: loss of structure in one part of the protein destabilizes other parts. The effects of heat on proteins are not readily predictable. The very heat-stable proteins of thermophilic bacteria have evolved to function at the temperature of hot springs (~100 0C). But the structures of these proteins often differ only slightly from those of homologous proteins derived from bacteria. How these small differences promote structural stability at high temperatures is not yet understood.

Proteins can be denatured by heat, extremes of pH, by certain miscible organic solvents such as alcohol or acetone, by certain solutes such as urea and guanidine hydrochloride, or by detergents.

Each of these denaturing agents represents a relatively mild treatment in the sense that no covalent bonds in the polypeptide chain are broken. Organic solvents, urea, and detergents act primarily by disrupting the hydrophobic interactions that make up the stable core of globular proteins; extremes of pH alter the net charge on the protein, causing electrostatic repulsion and the disruption of some hydrogen bonding. The denatured states obtained with these various treatments need not be equivalent

Amino Acid Sequence Determines

Tertiary Structure The tertiary structure of a globular protein is determined by its amino acid sequence. The most important proof of this came from experiments showing that denaturation

of some proteins is reversible. Certain globular proteins denatured by heat, extremes of pH, or denaturing reagents will regain their native structure and their biological activity if returned to conditions in which the native conformation is stable. This process is called renaturation.

A classic example is the denaturation and renaturation of ribonuclease. Purified ribonuclease can be completely denatured by exposure to a concentrated urea solution in the presence of a reducing agent. The reducing agent cleaves the four disulfide bonds to yield eight Cys residues, and the urea disrupts

unfolded, used to denatureribonuclease,andmercaptoethanol cleave the yieldeightCysresidues.

of the

Figure1.9.3Asimulatedfoldingpathway.Thefoldingpathway of a 36-residuesegment of the proteinvillin(anactin-binding protein found principally in the microvilliliningtheintestine) wassimulated by computer.Theprocessstartedwith the randomlycoiledpeptideand3,000 surroundingwater molecules in avirtual “water box ” Themolecularmotions of the peptideand the effects of the watermoleculesweretaken intoaccount in mapping the mostlikelypaths tothe final structureamong the countlessalternatives. The simulated folding took place in atheoreticaltimespan of 1 ms;however, the calculationrequiredhalfabillionintegrationsteps on two Craysupercomputers, each running for twomonths.

the stabilizing hydrophobic interactions, thus freeing the entire polypeptide from its folded conformation. Denaturation of ribonuclease is accompanied by a complete loss of catalytic activity. When the urea and the reducing agent are removed, the randomly coiled, denatured ribonuclease spontaneously refolds into its correct tertiary structure, with full restoration of its catalytic activity (Fig. 4 27). The refolding of ribonuclease is so accurate that the four intra chain disulfide bonds are re-formed in the same positions in the renatured molecule as in the native ribonuclease. The eight Cys residues could recombine at random to form up to four disulfide bonds in 105 different ways. In fact, an essentially random distribution of disulphide bonds is obtained when the disulfides are allowed to reform in the presence of denaturant, indicating that weak bonding interactions are required for correct positioning of disulfide bonds and assumption of the native conformation.

In living cells, proteins are assembled from amino acids at a very high rate. For example, E. coli cells can make a complete, biologically active protein molecule containing 100 amino acid residues in about 5 seconds at 37 such a polypeptide chain arrive at its native conformation? Let’s assume conservatively that each of the amino acid residues could take up 10 different conformations on average, giving 10100 different conformations for the polypeptide. Let’s also assume that the protein folds itself spontaneously by a random process in which it tries out all possible conformations around every single bond in its backbone until it finds its native, biologically active form. If each conformation were sampled in the shortest possible time (~10 13 molecular vibration), it would take about 10 all possible conformations. Thus protein folding cannot be a completely random, trialshortcuts. This problem is sometimes called paradox.

Figure1.9.4Thethermodynamics of afree-energy number of conformations,andhencethe large.Onlya intramolecular thenative

As folding thermodynamicpathdown of states increases the the native the free sides of the funnelrepresentsemistablefolding some cases,

The folding pathway of a large polypeptide chain is unquestionably complicated, and not all the principles that guide the process have been worked out. However, extensive study has led to the development of several. plausible models. In one, the folding process is envisioned as hierarchical. Local secondary structures form first. Certain amino acid sequences fold readily into helices or sheets guided by constraints we have reviewed in our discussion of secondary structure. This is followed by longer-range interactions between, say, two helices that come together to form stable super secondary structures. The process

Understanding Biochem

Defects in proteinfoldingmay be themolecularbasisforawiderange of human geneticdisorders. For example,cysticfibrosis is caused by defects in amembranebound proteincalled cystic fibrosis transmembraneconductance regulator(CFTR), whichacts as a channel forchlorideions.Themostcommoncysticfibrosis–causing mutation is thedeletion of aPheresidue at position 508 in CFTR,which causes improperproteinfolding.Many of thediseaserelatedmutations in collagen (p. 129) also cause defectivefolding. An improvedunderstanding of proteinfoldingmaylead to new therapiesforthese and manyotherdiseases

continues until complete domains form and the entire polypeptide is folded (Fig.1.9.3). In an alternative model, folding is initiated by a spontaneous collapse of the polypeptide into a compact state, mediated by hydrophobic interactions among nonpolar residues. The state resulting from this “hydrophobic collapse” may have a high content of secondary structure, but many amino acid side chains are not entirely fixed. The collapsed state is often referred to as a molten globule. Most proteins probably fold by a process that incorporates features of both models. Instead of following a single pathway, a population of peptide molecules

may take a variety of routes to the same end point, with the number of different partly folded conformational species decreasing as folding nears completion.

Thermodynamically, the folding process can be viewed as a kind of free-energy funnel (Fig. 1.9.4). The unfolded states are characterized by a high degree of conformational entropy and relatively high free energy. As folding proceeds, the narrowing of the funnel represents a decrease in the number of conformational species present. Small depressions along the sides of the free-energy funnel represent semi stable intermediates that can briefly slow the folding process. At the bottom of the funnel, an ensemble of folding intermediates has been reduced to a single native conformation (or one of a small set of native conformations).

Not all proteins fold spontaneously as they are synthesized in the cell. Folding for many proteins is facilitated by the action of specialized proteins. Molecular chaperones are proteins that interact with partially folded

or improperly folded polypeptides, facilitating correct folding pathways or providing microenvironments in which folding can occur. Two classes of molecular chaperones have been well studied. Both are found in organisms ranging from bacteria to humans. The first class, a family of proteins called Hsp70, generally have a molecular weight near 70,000 and are more abundant in cells stressed by elevated temperatures (hence, heat shock proteins of Mr 70,000, or Hsp70). Hsp70 proteins bind to regions of unfolded polypeptides that are rich in hydrophobic residues, preventing inappropriate aggregation.

These chaperones thus “protect” proteins that have been denatured by heat and peptides that are being synthesized (and are not yet folded). Hsp70 proteins also block the folding of certain proteins that must remain unfolded until they have been translocated across membranes.

Some chaperones also facilitate the quaternary assembly of oligomeric proteins. The Hsp70 proteins bind to and release polypeptides in a cycle that also involves several other proteins (including a class called Hsp40) and ATP hydrolysis. Figure 1.9.5 illustrates chaperone assisted folding as elucidated

Figure1.96 :Chaperonins in

GroEL (a member of theHsp60

formed by twoheptamericrings(eachsubunit

oneof theGroELpockets

10,000),

for the chaperones DnaK and Dna J in E. coli, homologs of the eukaryotic Hsp70 and Hsp40. DnaK and DnaJ were first identified as proteins required for in vitro replication of certain viral DNA molecules (hence the “DNA” designation).

The second class of chaperones is called chaperonins. These are elaborate protein complexes required for the folding of a number of cellular proteins that do not fold spontaneously. In E. coli an estimated 10% to 15% of cellular proteins require the resident chaperonin system, called GroEL/GroES, for folding under normal conditions (up to 30% require this assistance when the cells are heat stressed). These proteins first became known when they were found to be necessary for the growth of certain bacterial viruses (hence the

designation “Gro”). Unfolded proteins are bound within pockets in the GroEL complex, and the pockets are capped transiently by the GroES “lid” (Fig. 1.96). GroEL undergoes substantial conformational changes, coupled to ATP hydrolysis and the binding and release of GroES, which promote folding of the bound polypeptide. Although the structure of the GroEL/GroES chaperonin is known, many details of its mechanism of action remain unresolved.

Finally, the folding pathways of a number of proteins require two enzymes that catalyze isomerization reactions. Protein disulfide isomerase (PDI) is a widely distributed enzyme that catalyzes the interchange or shuffling of disulfide bonds until the bonds of the native conformation are formed. Among its functions, PDI catalyzes the elimination of folding intermediates with inappropriate disulfide cross-links. Peptide prolyl cistrans isomerase (PPI) catalyzes the interconversion of the cis and trans isomers of Pro peptide bonds, which can be a slow step in the folding of proteins that contain some Pro residue peptide bonds in the cis conformation. Protein folding is likely to be a more complex process in the densely packed cellular environment than in the test tube. More classes of proteins that facilitate protein folding may be discovered as the biochemical dissection of the folding process continues

1.9.2 Stability of Nucleic Acids

The role of DNA as a repository of genetic information depends in part on its inherent stability. The chemical transformations that do occur are generally very slow in the absence of an enzyme catalyst. The long-term storage of information without alteration is so important to a cell, however, that even very slow reactions that alter DNA structure can be physiologically significant.

Figure 1.9.7Heat denaturation of DNA. (a) The denaturation, or melting, curvesof two DNA specimens. The temperature at the midpoint of the transition (tm) is the melting point; it depends on pH and ionic strength and on the size and base composition of the DNA.

Processes such as carcinogenesis and aging may be intimately linked to slowly accumulating, irreversible alterations of DNA. Other, non-destructive alterations also occur and are essential to function, such as the strand separation that must precede DNA replication or transcription. In addition to providing insights into physiological processes, our understanding of nucleic acid chemistry has given us a powerful array of technologies that have applications in molecular biology, medicine, and forensic science. We now examine the chemical properties of DNA and some of these technologies.

Denaturation and renaturation

Solutions of carefully isolated, native DNA are highly viscous at pH 7.0 and room temperature (25oC). When such a solution is subjected to extremes of pH or to temperatures above 80 C, its viscosity

decreases sharply, indicating that the DNA has undergone a physical change. Just as heat and extremes of pH denature globular proteins, they also cause denaturation, or melting, of double-helical DNA. Disruption of the hydrogen bonds between paired bases and of base stacking causes unwinding of the double helix to form two single strands, completely separate from each other along the entire length or part of the length (partial denaturation) of the molecule. No covalent bonds in the DNA are broken (Fig.1.9.7).

Renaturation of a DNA molecule is a rapid one-step process, as long as a double-helical segment of a dozen or more residues still unites the two strands. When the temperature or pH is returned to the range in which most organisms live, the unwound segments of the two strands spontaneously rewind, or anneal, to yield the intact duplex (Fig. 1.9.7). However, if the two strands are completely separated, renaturation occurs in two steps. In the first, relatively slow step, the two strands “find” each other by random collisions and form a short segment of complementary double helix. The second step is much faster: the remaining unpaired bases successively come into register as base pairs, and the two strands “zipper” themselves together to form the double helix. The close interaction between stacked bases in a nucleic acid has the effect of decreasing its absorption of UV light relative to that of a solution with the same concentration of free nucleotides, and the absorption is decreased further when two complementary nucleic acids strands are paired. This is called the hypochromic effect. Denaturation of a double-stranded nucleic acid produces the opposite result: an increase in absorption called the Hyperchromic effect.

commonnucleotides.The molarextinctioncoefficient nm and pH corresponding as the nucleotides,a absorption

The transition from double-stranded DNA to the single-stranded, denatured form can thus be detected by monitoring the absorption of UV light. Viral or bacterial DNA molecules in solution denature when they are heated slowly (Fig. 1.9.7). Each species of DNA has a characteristic denaturation temperature, or melting point (tm): the higher its content of GqC base pairs, the higher the melting point of the DNA. This is because GqC base pairs, with three hydrogen bonds, require more heat energy to dissociate than AUT base pairs. Careful determination of the melting point of a DNA specimen, under fixed conditions of pH and ionic strength, can yield an estimate of its base composition. If denaturation conditions are carefully controlled, regions that are rich in AUT base pairs will specifically denature while most of the DNA remains double stranded.

Strand separation of DNA must occur in vivo during processes such as DNA replication and transcription. As we shall see, the DNA sites where these processes are initiated are often rich in AUT base pairs. Duplexes of two RNA strands or of one RNA strand and one DNA strand (RNA-DNA hybrids) can also be denatured. Notably, RNA duplexes are more stable than DNA duplexes. At neutral pH, denaturation of a double helical RNA often requires temperatures 20oC or higher than those required for denaturation of a DNA molecule with a comparable sequence. The stability of an RNA-DNA hybrid is generally intermediate between that of RNA and that of DNA. The physical basis for these differences in thermal stability is not known.

are very nearly planar

large absorbance

Der Waals

hydrogen bond

Free pyrimidine and purines are weakly basic compounds and are thus called bases. They have a variety of chemical properties that affect the structure, and ultimately the function, of nucleic acids. The purines and pyrimidines common in DNA and RNA are highly conjugated molecules, a property with important consequences for the structure, electron distribution, and light absorption of nucleic acids. Resonance among atoms in the ring gives most of the bonds partial double-bond character. One result is that pyrimidines are planar molecules; purines are very nearly planar, with a slight pucker. Free pyrimidine and purine bases may exist in two or more tautomeric forms depending on the pH. As a result of resonance, all nucleotide bases absorb UV light, and nucleic acids are characterized by a strong absorption at wavelengths near 260 nm (Fig. 1.9.9). The purine and pyrimidine bases are hydrophobic and relatively insoluble in water at the near-neutral pH of the cell. At acidic or alkaline pH the bases become charged and their solubility in water increases. Hydrophobic stacking interactions in which two or more bases are positioned with the planes of their rings parallel (like a stack of coins) are one of two important modes of interaction between bases in nucleic acids. The stacking also involves a combination of van der Waals and dipole-dipole interactions between the bases. Base stacking helps to minimize contact of the bases with water, and base-stacking interactions are very important in stabilizing the three-dimensional structure of nucleic acids.

Trans esterification reaction

RNA , bur not DNA can undergo the trans esterification reaction . This property is attributed to the presence of 2’OH group on the ribose sugar, which is absent in DNA. The following figure 1.9.9 shows the mechanism of the reaction.

Figure 1.9.9 Trans-esterification reaction. This is the first step in the guanosinemolecule acts as

Critical thinking Questions

1. Supposeyouwant bacterialcells.Whichradioactivemoleculewouldyouadd

2. RNA is

3. Theaminoacidcoded by codonGGGcouldnot be deciphered in thesameway as UUU, CCC or AAA.Why is polyG an ineffectivetemplate?

4. Griffithused heat-killedS.Pneumococci to transformRmutants.Studiesyearslater showedthatdsDNA is neededforefficienttransformationandthathightemperaturemelt theDNAdoublehelix.Whywere Griffith’s experimentsneverthelesssuccessful?

5. In designing of primers,the Tm of eachprimershouldapproximately be thesame.What is thebasis of thisrequirement?

1.10 Metabolism of carbohydrates, lipids, amino acids, nucleotides and vitamins.

1.10.1 Carbohydrates

Snapshot

8. The metabolism of carbohydrates is dominated by glucose because this sugar is an important fuel molecule in most organisms. If cellular energy reserves are low, glucose is degraded by the glycolytic pathway. Glucose molecules that are not required for immediate energy production are stored as either glycogen (in animals) or starch (in plants).

9. During glycolysis, glucose is phosphorylated and cleaved to form two molecules of glyceraldehyde-3-phosphate. Each glyceraldehyde-3-phosphate is then converted to a molecule of pyruvate. A small amount of energy is captured in two molecules each of ATP and NADH. In anaerobic organisms, pyruvate is converted to waste products. During this process, NAD_ is regenerated so that glycolysis can continue. In the presence of O2, aerobic organisms convert pyruvate to acetyl-CoA and then to CO2 and H2O. Glycolysis is controlled primarily by allosteric regulation of three

10. During gluconeogenesis, molecules of glucose are synthesized from noncarbohydrate precursors (lactate, pyruvate,

11. is largely the reverse of glycolysis. The three irreversible glycolytic reactions (the synthesis of pyruvate, the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate, and the formation of glucose from glucose-6-phosphate) are

12. The pentose phosphate pathway, in which glucose-6-phosphate is oxidized, occurs in two phases. In the oxidative phase, two molecules of NADPH are produced as glucose-6- phosphate is converted to ribulose-5-phosphate. In the noxidative phase, ribose-5-phosphate and other sugars are synthesized. If cells need more NADPH than ribose-5phosphate, a component of nucleotides and the nucleic acids, then metabolites of the nonoxidative phase are converted

13. Several sugars other than glucose are important in vertebrate carbohydrate metabolism. These include fructose,

14.

-glucose pyrophosphorylase catalyzes the formation of UDP-glucose from glucose-1-phosphate and UTP. Glucose-6-phosphate is converted to glucose-1-phosphate by phosphoglucomutase. Glycogen synthesis requires two enzymes: glycogen synthase and branching enzyme. Glycogen degradation requires glycogen phosphorylase and debranching enzyme. The balance between glycogenesis (glycogen synthesis) and glycogenolysis (glycogen breakdown) is carefully regulated by several

Living cells are in a state of ceaseless activity. To maintain its “life,” each cell depends on highly coordinated biochemical reactions. Carbohydrates are an important source of the energy that drives these reactions.

During , an ancient pathway found in almost all organisms, a small amount of energy is captured as a glucose molecule is converted to two molecules of pyruvate.

 Glycogen, a storage form of glucose in vertebrates, is synthesized by when glucose levels are high and degraded by when glucose is in short supply. Glucose can also be synthesized from noncarbohydrate precursors by reactions referred to as gluconeogenesis

The pentose phosphate pathway enables cells to convert glucose-6-phosphate, a derivative of glucose, to ribose- 5-phosphate (the sugar used to synthesize nucleotides and nucleic acids) and other types of monosaccharides (Figure 1.10.1).

NADPH, an important cellular reducing agent, is also produced by this pathway.

Glycolysis

Glycolysis, occurs, at least in part, in almost every living cell. This series of reactions is believed to be among the oldest of all the biochemical pathways.

Both the enzymes and the number and mechanisms of the steps in the pathway are highly conserved in prokaryotes and eukaryotes.

Also, glycolysis is an anaerobic process, which would have been necessary in the oxygen-poor atmosphere of pre-eukaryotic Earth.

In glycolysis, also referred to as the , each glucose molecule is split and converted to two three-carbon units (pyruvate).

During this process several carbon atoms are oxidized. The small amount of energy captured during glycolytic reactions (about 5% of the total available) is stored temporarily in two molecules each of ATP and NADH (the reduced form of the coenzyme NAD

An ELISA(enzyme-linkedimmunosorbentassay)allowsforrapidscreening and quantification of thepresence of an antigen in asample.Proteins in asampleare adsorbed to an inertsurface,usuallya 96-wellpolystyreneplate.Thesurface is washedwithasolution of an inexpensive nonspecific protein(oftencaseinfrom nonfatdrymilkpowder) to blockproteinsintroduced in subsequent stepsfromalso adsorbing to thesesurfaces.Thesurface is thentreatedwithasolutioncontaining theprimaryantibody—an antibodyagainsttheprotein of interest.Unboundantibody is washedaway and thesurface is treatedwithasolutioncontainingantibodies againsttheprimaryantibody.Thesesecondaryantibodieshave been linked to an enzymethatcatalyzesareactionthatformsacoloredproduct.After unbound secondary antibody is washedaway,thesubstrate of theantibody-linkedenzyme is added.Productformation(monitored as colorintensity) is proportional to the concentration of theprotein of interest in thesample.

pyruvate may be converted to waste products such as ethanol, lactic acid, acetic acid, and similar molecules.

Using oxygen as a terminal electron acceptor, aerobic organisms such as animals and plants completely oxidize pyruvate to form CO2 and H2O in an elaborate stepwise mechanism known as aerobic respiration.

Glycolysis, which consists of 10 reactions, occurs in two stages:

3. Glucose is phosphorylated twice and cleaved to form two molecules of glyceraldehyde-3phosphate (G-3-P). The two ATP molecules consumed during this stage are like an investment, because this stage creates the actual substrates for oxidation in a form that is trapped inside the cell.

4. Glyceraldehyde-3-phosphate is converted to pyruvate. Four ATP and two NADH molecules are produced. Because two ATP were consumed in stage 1, the net production of ATP per glucose

The glycolytic pathway can be summed up in the following equation:

The Reactions of the Glycolytic Pathway

The 10 reactions of the glycolytic pathway are as follows.

11. Synthesis of glucose-6-

Immediately after entering a cell, glucose and other sugar molecules are phosphorylated. Phosphorylation prevents transport of glucose out of the cell and increases the reactivity of

Several enzymes, called the hexokinases, catalyze the phosphorylation of hexoses in all cells complexes

Under intracellular conditions the reaction is irreversible; that is, the enzyme has no ability to retain or accommodate the product of the reaction in its active site, regardless of the

12. Conversion of glucose-6-



During reaction 2 of glycolysis, the open chain form of the aldose glucose-6-phosphate is converted to the open chain form of the ketose fructose-6-phosphate byphosphoglucose isomerase (PGI) in a readily reversible reaction:

13. The phosphorylation of fructose-6-phosphate. Phosphofructokinase-1 (PFK-1) irreversibly catalyzes the phosphorylation of fructose-6-phosphate to form fructose-1,6-bisphosphate:

The PFK-1-catalyzed reaction is irreversible under cellular conditions. It is, therefore, the first

Unlike glucose-6- phosphate and fructose-6-phosphate, the substrate and product, respectively, of the previous reaction, fructose-1,6-bisphosphate cannot be diverted into other pathways.

First of all, because ATP is used as the phosphorylating agent, the reaction proceeds with a large

After fructose-1,6-bisphosphate has been synthesized, the cell is committed to glycolysis.

Because fructose-1,6-bisphosphate eventually splits into two trioses, another purpose for phosphorylation is to prevent any later product from diffusing out of the cell because charged

14. Cleavage of fructose-1,6-bisphosphate.

Stage 1 of glycolysis ends with the cleavage of fructose-1,6-bisphosphate into two three-carbon molecules: glyceraldehyde-3-phosphate (G-3-P) and dihydroxyacetone phosphate (DHAP).

Although the cleavage of fructose-1,6-bisphosphate is thermodynamically unfavorable, the reaction proceeds because the products are rapidly removed.

15. The interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.

Of the two products of the aldolase reaction, only G-3-P serves as a substrate for the next reaction in glycolysis.

To prevent the loss of the other three-carbon unit from the glycolytic pathway, triose phosphate isomerase catalyzes the reversible conversion of DHAP to G-3-P:

After this reaction, the original molecule of glucose has been converted to two molecules of G-3-P.

16. Oxidation of glyceraldehyde-3-

During reaction 6 of glycolysis, G-3-P undergoes oxidation and phosphorylation. The product, glycerate-1,3-bisphosphate, contains a high-energy phosphoanhydride bond, which may be used in the next reaction to generate ATP:

This complex process is catalyzed by glyceraldehyde-3-phosphate dehydrogenase, a tetramer composed of four identical subunits.

Each subunit contains one binding site for G-3-P and another for NAD , an oxidizing ogent. As the enzyme forms a covalent thioester bond with the substrate, a hydride ion (H: ) is transferred to NAD+ in the active site.

NADH, the reduced form of NAD , then leaves the active site and is replaced by an incoming NAD+ The acyl enzyme adduct is attacked by inorganic phosphate and the product leaves the active site.

17. Phosphoryl group transfer.

In this reaction ATP is synthesized as phosphoglycerate kinase catalyzes the transfer of the highenergy phosphoryl group of glycerate-1,3-bisphosphate to ADP:

Reaction 7 is an example of a substrate-level phosphorylation. Because the synthesis of ATP is endergonic, it requires an energy source. In substratelevel phosphorylations, ATP is produced by the transfer of a phosphoryl group from a substrate with a high phosphoryl transfer potential (glycerate1,3-bisphosphate) to produce a compound with a lower transfer potential (ATP).

Because two molecules of glycerate-1,3-bisphosphate are formed for every glucose molecule, this reaction produces two ATP molecules, and the investment of phosphate bond energy is recovered.

ATP synthesis later in the pathway represents a net gain.

18. The interconversion of 3-phosphoglycerate and 2-phosphoglycerate.

Glycerate-3-phosphate has a low phosphoryl group transfer potential. As such, it is a poor candidate for further ATP synthesis.

Cells convert glycerate-3-phosphate with its energy-poor phosphate ester to phosphoenolpyruvate (PEP), which has an exceptionally high phosphoryl group transfer potential.

In the first step in this conversion (reaction 8), phosphoglycerate mutase catalyzes the conversion of a C-3 phosphorylated compound to a C-2 phosphorylated compound through a two-step addition/elimination cycle.

19. Dehydration of 2-phosphoglycerate.

Enolase catalyzes the dehydration of glycerate-2-phosphate to form PEP:

PEP has a higher phosphoryl group transfer potential than does glycerate-

2- phosphate because it contains an enol-phosphate group instead of a simple phosphate ester. The reason for this difference is made apparent in the next reaction.

Aldehydes and ketones have two isomeric forms. The enol form contains a carbon-carbon double bond and a hydroxyl group.

Enols exist in equilibrium with the more stable carbonyl-containing keto form. The interconversion of keto and enol forms, also called tautomers, is referred to as tautomerization:

This tautomerization is restricted by the presence of the phosphate group, as is the resonance stabilization of the free phosphate ion. As a result, phosphoryl transfer to ADP in reaction 10 is highly favored.

20. Synthesis of pyruvate.

In the final reaction of glycolysis, pyruvate kinase catalyzes the transfer of a phosphoryl group from PEP to ADP. Two molecules of ATP are formed for each molecule of glucose.

PEP is irreversibly converted to pyruvate because in this reaction the transfer of a phosphoryl group from a molecule with a high transfer potential to one with a lower transfer potential there is an exceptionally large free energy loss.

This energy loss is associated with the spontaneous conversion (tautomerization) of the enol form of pyruvate to the more stable keto form. The 10 reactions of glycolysis are illustrated in (Figure 1.10.2

The Fates of Pyruvate

In terms of energy, the result of glycolysis is the production of two ATPs and two NADHs per molecule of glucose.

Pyruvate, the other product of glycolysis, is still an energy-rich molecule, which can yield a substantial amount of ATP.

of Pyruvate

Whether or not further energy can be produced, however, depends on the cell type and the availability of oxygen.

Under aerobic conditions, most cells in the body convert pyruvate into acetyl-CoA, the entry-level substrate for the citric acid cycle, an amphibolic pathway that completely oxidizes the two acetyl carbonstoform CO2 and the reducedmolecules NADH and FADH2 . (An amphibolic pathway functions in both anabolic and catabolic processes.)

The electron transport system, a series of oxidation- reduction reactions, transfers electrons from NADH and FADH2 to O2 to form water.



The energy that is released during electron transport is coupled to a mechanism that synthesizes ATP. Under anaerobic conditions, further oxidation of pyruvate is impeded.

A number of cells and organisms compensate by converting ng this molecule to a more reduced organic compound and regenerating the NAD+ required for glycolysis to continue (Figure 1.10.3) that the hydride ion acceptor molecule NAD+ is a cosubstrate in the reaction catalyzed by glyceraldehyde3-phosphate dehydrogenase.)

This process of NAD+ regeneration is referred to as fermentation. Muscle cells, red blood cells, and certain bacterial species (e.g., Lactobacillus) produce NAD+ by transforming pyruvate into lactate:

In rapidly contracting muscle cells, the demand for energy is high. After the O supply is depleted, lactic acid fermentation provides sufficient NAD+ to allow glycolysis (with its low level of ATP production) to continue for a short time.

This process, called , is used commercially to produce wine, beer, and bread. The Energetics of Glycolysis





During glycolysis, the energy released as glucose is broken down to pyruvate is coupled to the phosphorylation of ADP with a net yield of 2 ATP.

Regulation of Glycolysis



The rate at which the glycolytic pathway operates in a cell is directly controlled primarily by the kinetic properties of its hexokinase isoenzymes and the allosteric regulation of the enzymes that catalyze the three irreversible reactions: hexokinase, PFK-1, and pyruvate kinase.

Gluconeogenesis



Gluconeogenesis, the formation of new glucose molecules from noncarbohydrate precursors, occurs primarily in the liver.

Precursor molecules include lactate, pyruvate, glycer -keto acids (molecules derived from amino acids).

Under certain conditions (i.e., metabolic acidosis or starvation) the kidney can make small amounts of new glucose.

Between meals adequate blood glucose levels are maintained by the hydrolysis of liver glycogen.

When liver glycogen is depleted (e.g., owing to prolonged fasting or vigorous exercise), the gluconeogenesis pathway provides the body with adequate glucose. Brain and red blood cells rely exclusively on glucose as their energy source.

Gluconeogenesis Reactions

The reaction sequence in gluconeogenesis is largely the reverse of glycolysis. Recall, however, that three glycolytic reactions (the reactions catalyzed by hexokinase, PFK-1, and pyruvate kinase) are irreversible. In gluconeogenesis, alternate reactions catalyzed by different enzymes are used to bypass these obstacles. The reactions unique to gluconeogenesis are listed next.

The entire gluconeogenic pathway and its relationship to glycolysis are illustrated in Figure 1.10.4. The bypass reactions of gluconeogenesis are as follows:

The transfer of CO to form the product OAA is mediated by the coenzyme biotin, which is covalently bound within the enzyme’s active site. OAA is then decarboxylated and phosphorylated by PEP car boxykinase in a reaction driven by the hydrolysis of guanosine triphosphate (GTP):

PEP carboxykinase is found within the mitochondria of some species and in the cytoplasm of others. In humans this enzymatic activity is found in both compartments. Because the inner mitochondrial membrane is impermeable to OAA, cells that lack mitochondrial PEP carboxykinase transfer OAA into the cytoplasm by using the malate shuttle. In this process, OAA is converted into malate by mitochondrial malate dehydrogenase. After the transport of malate across mitochondrial membrane, the reverse reaction (to form OAA and NADH) is catalyzed by cytoplasmic malate dehydrogenase. The malate shuttle allows gluconeogenesis to continue because it provides the NADH required for the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase.

2. Conversion of fructose-1,6-bisphosphate to fructose-6-phosphate.

 The irreversible PFK-1 catalyzed reaction in glycolysis is bypassed by fructose-1,6-bisphosphatase:

This exergonic reaction is also irreversible under cellular conditions. ATP is not regenerated, and inorganic phosphate (Pi) is also produced.

Fructose-1,6-bisphosphatase is an allosteric enzyme. Its activity is stimulated by citrate and inhibited by AMP and fructose- 2,63. Formation of glucose from glucose-6-phosphate.

Glucose-6-phosphatase, found only in liver and kidney, catalyzes the irreversible hydrolysis of glucose-6-phosphate to form glucose and Pi.

Glucose is subsequently released into the blood. Each of the foregoing reactions is matched by an opposing irreversible reaction in glycolysis.

Each set of such paired reactions is referred to as a substrate cycle. Because they are coordinately regulated (an activator of the enzyme catalyzing the forward reaction serves as an inhibitor of the enzyme catalyzing the reverse reaction), very little energy is wasted, even though both enzymes may be operating at some level at the same time.

Flux control (regulation of the flow of substrate and removal of product) is more effective if transient accumulation of product is funneled back through the cycle.

The catalytic velocity of the forward enzyme will remain high if the concentration of the substrate is maximized. The gain in catalytic efficiency more than makes up for the small energy loss in recycling the product.

Gluconeogenesis is an energy-consuming process. Instead of generating ATP (as in glycolysis), gluconeogenesis requires the hydrolysis of six high energy phosphate bonds. Gluconeogenesis Substrates



As previously mentioned, several metabolites are gluconeogenic precursors. Three of the most important substrates are described briefly. Lactate is released by red blood cells and other cells that lack mitochondria or

have low oxygen concentrations. In the Cori cycle, lactate is released by skeletal muscle during exercise ( ). After lactate is transferred to the liver, it is reconverted to pyruvate by lactate dehydrogenase and then to glucose by gluconeogenesis.



Glycerol, a product of fat metabolism in adipose tissue, is transported to the liver in the blood and then converted to glycerol-3-phosphate by glycerol kinases.

 Oxidation of glycerol-3-phosphate to form DHAP occurs when cytoplasm NAD+ concentration is relatively high.

Figure1.10.5.TheCoriCycle.Duringstrenuous exercise,lactate is producedanaerobically in musclecells.Afterpassingthroughblood the

Of all the amino acids that can be converted to glycolytic intermediates (molecules referred to as glucogenic), alanine is perhaps the most important.

exercising muscle produces large quantities of pyruvate, some of these molecules are converted to alanine by a transamination reaction involving glutamate: After it has been transported to the liver, alanine is reconverted to pyruvate and then to glucose.

Gluconeogenesis Regulation

As with other metabolic pathways, the rate of gluconeogenesis is affected primarily by substrate availability, allosteric effectors, and hormones.

Not surprisingly, gluconeogenesis is stimulated by high concentrations of lactate, glycerol,and amino acids. A high-fat diet, starvation, and prolonged fasting make large quantities of these molecules available.

The four key enzymes in gluconeogenesis (pyruvate carboxylase, PEP carboxykinase, fructose1,6-bisphosphatase, and glucose-6-phosphatase) are affected to varying degrees by allosteric modulators.

For example, fructose-1,6bisphosphatase is activated by citrate and inhibited by AMP and fructose-2,6-bisphosphate. Acetyl-CoA activates pyruvate carboxylase. (The concentration of acetyl-CoA, a product of fatty acid degradation, is especially high during starvation.)

Figure Figure 1.10.6 provide an overview of the allosteric

As with other biochemical

by altering the concentrations of allosteric effectors and the key rate-determining

As mentioned previously, glucagon depresses the synthesis of fructose- 2,6-bisphosphate, which releases the inhibition of fructose-1,6-bisphosphatase, and inactivates the glycolytic enzyme

Hormones also influence gluconeogenesis by altering enzyme synthesis.

For example, the synthesis of gluconeogenic enzymes is stimulated by cortisol, a steroid hormone produced in the cortex of the adrenal gland that facilitates the body’s adaptation

nally, insulin action leads to the synthesis of new molecules of glucokinase, PFK-1 (SREBP1cinduced), and PFK-2 (glycolysis favored). Insulin also depresses the synthesis (also via SREBP1c) of PEP carboxykinase, fructose- 1,6-bisphosphatase, and glucose-6-phosphatase. Glucagon action leads to the synthesis of additional molecules of PEP carboxykinase, fructose-

The hormones that regulate glycolyses and gluconeogenesis alter the phosphorylation state of

-lactamases,whichprovidebacteriawithabulletproofvest.Abeta-lactamaseforms atemporarycovalentadductwiththecarboxylgroup of the opened beta-lactamring, which is immediatelyhydrolyzed,regeneratingactiveenzyme.Oneapproach to circumventingantibioticresistance of thistype is to synthesizepenicillinanalogs, suchas methicillin,thatare poor substratesfor betaactamases.Anotherapproach is to administeralongwithantibioticsabeta-lactamaseinhibitorsuch as clavulanate or sulbactam.

-layingsites.Plants dungor

The key point to remember is that insulin and glucagon have opposing effects on carbohydrate metabolism.

The direction of metabolite flux, (i.e., whether either glycolysis or gluconeogenesis is active) is largely determined by the ratio of insulin to glucagon.

Figure1.10.6.AllostericRegulation of Glycolysis and GluconeogenesisThekeyenzymes in glycolysis and gluconeogenesisareregulated by allostericeffectors.Activator,+; inhibitor,-.



After a carbohydrate meal, the insulin/glucagon ratio is high and glycolysis in the liver predominates over gluconeogenesis.



After a period of fasting or following a high-fat, low-carbohydrate meal, the insulin/glucagon ratio is low and gluconeogenesis in the liver predominates over glycolysis.



The availability of ATP is the second important regulator in the reciprocal control of glycolysis and gluconeogenesis in that high levels of AMP, the low-energy hydrolysis product of ATP, increase the flux through glycolysis at the expense of gluconeogenesis, and low levels of AMP increase the flux through gluconeogenesis at the expense of glycolysis.



Although control at the PFK-1/fructose-1,6-bisphosphatase cycle would appear to be sufficient for this pathway, control at the pyruvate kinase step is key because it permits the maximal retention of PEP, a molecule with a very high phosphate transfer potential.

The Pentose Phosphate Pathway



The pentose phosphate pathway is an alternative metabolic pathway for glucose oxidation in which no ATP is generated.



Its principal products are NADPH, a reducing agent required in several anabolic processes, and ribose-5-phosphate, a structural component of nucleotides and nucleic acids.



The pentose phosphate pathway occurs in the cytoplasm in two phases: oxidative and nonoxidative.



In the oxidative phase of the pathway, the conversion of glucose-6-phosphate to ribulose-5phosphate is accompanied by the production of two molecules of NADPH.



The nonoxidative phase involves the isomerization and condensation of a number of different sugar molecules.



Three intermediates in this process that are useful in other pathways are ribose-5-phosphate, fructose-6-phosphate, and glyceraldehyde-3-phosphate.



The oxidative phase of the pentose phosphate pathway consists of three reactions (Figure 1.10.7).



In the first reaction, glucose-6-phosphate dehydrogenase (G-6-PD) catalyzes the oxidation of glucose-6-phosphate.



6-Phosphogluconolactone and NADPH are products in this reaction. 6-Phospho-D-glucono-δ-lactone is then hydrolyzed to produce 6-phospho-D-gluconate. A second molecule of NADPH is produced during the oxidative decarboxylation of 6-phosphogluconate, a reaction that yields ribulose-5phosphate.



A substantial amount of the NADPH required for reductive processes (i.e., lipid biosynthesis) is supplied by these reactions.



For this reason this pathway is most active in cells in which relatively large amounts of lipids are synthesized, (e.g., adipose tissue, adrenal cortex, mammary glands, and the liver). NADPH is also a powerful antioxidant. Consequently, the oxidative phase of the pentose phosphate pathway is also quite active in cells that are at high risk for oxidative damage, such as red blood cells.



The nonoxidative phase of the pathway begins with the conversion of ribulose-5-phosphate to ribose5-phosphate by ribulose-5-phosphate isomerase or to xylulose-5-phosphate by ribulose-5-phosphate epimerase.



During the remaining reactions of the pathway (Figure 1.10.8), transketolase and transaldolase catalyze the interconversions of trioses, pentoses, and hexoses.

Transketolase is a TPP requiring enzyme that transfers two-carbon units from a ketose to an aldose. (TPP,thiamine pyrophosphate, is the coenzyme form of thiamine, also known as vitamin B).

Transketolase catalyzes two reactions. In the first reaction, the enzyme transfers a two-carbon unit from xylulose-5-phosphate to ribose-5-phosphate, yielding glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate.



In the second transketolase-catalyzed reaction, a two-carbon unit from another xylulose-5-phosphate molecule is transferred to erythrose-4-phosphate to form a second molecule of glyceraldehyde-3phosphate and fructose-6-phosphate.

Figure1.10.9.CarbohydrateMetabolism:Glycolysis and thePentosePhosphatePathway If thecellrequiresmore NADPHthanribosemolecules, it canchanneltheproducts of thenonoxidativephase of thepentosephosphatepathway intoglycolysis. As thisoverview of thetwopathwaysillustrates,excessribulose-5-phosphatecan be convertedintothe glycolyticintermediatesfructose-6-phosphate and glyceraldehyde-3-phosphate.

Transaldolase transfers three-carbon units from a ketose to an aldose. In the reaction catalyzed by transaldolase, a three- carbon unit is transferred from sedoheptulose-7-phosphate to glyceraldehyde3-phosphate.

The products formed are fructose-6-phosphate and erythrose-4-phosphate. The result of the nonoxidative phase of the pathway is the synthesis of ribose-5-phosphate and the glycolytic intermediates glyceraldehyde-3- phosphate and fructose-6-phosphate.

When pentose sugars are not required for biosynthetic reactions, the metabolites in the nonoxidative portion of the pathway are converted into glycolytic intermediates that can then be further degraded to generate energy or converted into precursor molecules for biosynthetic processes (Figure 1.10.9).

For this reason the pentose phosphate pathway is also referred to as the hexose monophosphate shunt.

In plants, the pentose phosphate pathway is involved in the synthesis of glucose during the dark reactions of photosynthesis.

The pentose phosphate pathway is regulated to meet the cell’s momentby-moment requirements for NADPH and ribose-5-phosphate.

The oxidative phase is very active in cells such as red blood cells or hepatocytes in which demand for NADPH is high. In contrast, the oxidative phase is virtually absent in cells (e.g., muscle cells) that synthesize little or no lipid. (Lipid synthesis is a major consumer of NADPH.)

G-6-PD catalyzes a key regulatory step in the pentose phosphate pathway. Its activity is inhibited by NADPH and stimulated by GSSG, the oxidized form of glutathione, an important cellular antioxidant and glucose-6-phosphate.

In addition, diets high in carbohydrate increase the synthesis of both G-6-PD and phosphogluconate dehydrogenase.

Metabolism of Other Important Sugars

Several sugars other than glucose are important in vertebrates. The most notable of these are fructose, galactose, and mannose.

Besides glucose, these molecules are the most common sugars found in oligosaccharides and polysaccharides.

They are also energy sources. The reactions by which these sugars are converted into glycolytic intermediates are illustrated in (

The metabolism of fructose, an important component of the human diet, is discussed. Fructose Metabolism

Dietary sources of fructose include fruit, honey, sucrose, and high-fructose corn syrup, an inexpensive sweetener used in a wide variety of processed foods and beverages.

Fructose, second only to glucose as a source of carbohydrate in the modern human diet, can enter the glycolytic pathway by two routes. In the liver, fructose is converted to fructose-1-phosphate by fructokinase:

When fructose-1-phosphate enters the glycolytic pathway, it is first split into dihydroxyacetone phosphate (DHAP) and glyceraldehyde by fructose-1-phosphate aldolase.

DHAP is then converted to glyceraldehyde-3-phosphate by triose phosphate isomerase.

Glyceraldehyde-3-phosphate is generated from glyceraldehydes and ATP by glyceraldehyde kinase.

The conversion of fructose-1-phosphate into glycolytic intermediates bypasses two regulatory steps (the reactions catalyzed by hexokinase and PFK-1); thusin comparison to glucose, the entrance of fructose into the glycolytic pathway is essentially unregulated.

In muscle and adipose tissue, fructose is converted to the glycolytic intermediate fructose-6phosphate by hexokinase. Because the hexokinases have a low affinity for fructose, this reaction is of minor importance unless fructose consumption is exceptionally high.

Figure1.10

by tworoutes.Fructokinase in livercellsconvertsfructose to fructose-1-phosphatethat is thensplitintoDHAP and glyceraldehyde. In muscle and adiposetissue, fructose is phosphorylated by hexokinase to formtheglycolyticintermediatefructose-6-phosphate.Galactose is convertedinto galactose-1-phosphate,whichthenreactswithUDP-glucose to formUDP-galactose.UDP-galactose is converted to itsepimer,UDPglucose,thesubstrateforglycogensynthesis.Mannose is phosphorylated by hexokinase to formmannose-6-phosphate,which is then isomerized to fructose-6-phosphate.

Glycogen Metabolism

Glycogen is the storage form of glucose. The synthesis and degradation of glycogen are carefully regulated so that sufficient glucose is available for the body’s energy needs. Both glycogenesis and glycogenolysis are controlled primarily by three hormones: insulin, glucagon, and epinephrine.

Glycogenesis

Glycogen synthesis occurs after a meal, when blood glucose levels are high. It has long been recognized that the consumption of a carbohydrate meal is followed promptly by liver glycogenesis. The synthesis of glycogen from glucose-6-phosphate involves the following set of reactions.

1. Synthesis of glucose-1-phosphate.

Glucose-6-phosphate is reversibly converted to glucose-1-phosphate by phosphoglucomutase, an enzyme that contains a phosphoryl group attached to a reactive serine residue:

The enzyme’s phosphoryl group is transferred to glucose-6-phosphate, forming glucose- 1,6bisphosphate. As glucose-1-phosphate forms, the phosphoryl group attached to C-6 is transferred to the enzyme’s serine residue.

2. Synthesis of UDP-glucose.

Glycosidic bond formation is an endergonic process. Derivatizing the sugar with a good leaving group provides the driving force for most sugar transfer reactions.

For this reason, sugar-nucleotide synthesis is a common reaction preceding sugar transfer and polymerization processes.

Uridine diphosphate glucose (UDP-glucose) is more reactive than glucose and is held more securely in the active site of the enzymes catalyzing transfer reactions (referred to as a group as glycosyl transferases).

Because UDP-glucose contains two phosphoryl bonds, it is a highly reactive molecule. Formation of UDP

However, the reaction is driven to completion because pyrophosphate (PPi) is immediately and irreversibly hydrolyzed by pyrophosphatase with a large loss of free energy. Synthesis of glycogen from UDP-glucose.

The formation of glycogen from UDP-glucose requires two enzymes: (a) glycogen synthase, which catalyzes the transfer of the glucosyl group of UDP-glucose to the nonreducing ends of glycogen (Figure 1.10.11a), and (b) amylo-α(1,4 →1,6)-glucosyl transferase (branching enzyme), which creates the α(1,6) linkages for branches in the molecule (Figure 1.10.11b).

Figure1.10 11.GlycogenDegradationGlycogenphosphorylasecatalyzestheremoval of glucoseresidues fromthenonreducingends of aglycogenchain to yieldglucose-1-phosphate. In thisillustrationoneglucose residue is removedfromeach of twononreducing ends.Removal of glucoseresiduescontinuesuntilthereare fourresidues at abranchpoint.

Glycogen synthesis requires a preexisting tetrasaccharide composed of four α (1, 4)-linked glucosyl residues.

The first of these residues is linked to a specific tyrosine residue in a “primer” protein called glycogenin.

The glycogen chain is then extended by glycogen synthase and branching enzyme. Large glycogen granules, each consisting of a single highly branched glycogen molecule, can be observed in the cytoplasm of liver and muscle cells of well-fed animals. The enzymes responsible for glycogen synthesis and degradation coat each granule’s surface.

Glycogenolysis

Glycogen degradation requires the following two reactions.

1. Removal of glucose from the nonreducing ends of glycogen. Glycogen phosphorylase uses inorganic phosphate ( 1, 4) linkages on the outer branches of glycogen to yield glucose-1phosphate. Glycogen phosphorylase stops when it comes within four glucose residues of a branch point ( ). (A glycogen molecule that has been degraded to its branch points is called a limit dextrin.)

Figure1.10.12.GlycogenDegradationGlycogenphosphorylasecatalyzestheremoval of glucoseresidues fromthenonreducingends of aglycogenchain to yieldglucose-1-phosphate. In thisillustrationoneglucose residue is removedfromeach of twononreducingends.Removal of glucoseresiduescontinuesuntilthere arefourresidues at abranchpoint.

2. Hydrolysis of the a(1,6) glycosidic bonds at branch points of glycogen. Amylo-α(1,6)-glucosidase, also called debranching enzyme, begins the removal of α(1,6) branch points by transferring the outer three of the four glucose residues attached to the branch point to a nearby nonreducing end. It then removes

the single glucose residue attached at each branch point. The product of this latter reaction is free glucose.

3. Glucose-1-phosphate, the major product of glycogenolysis, is diverted to glycolysis in muscle cells to generate energy for muscle contraction. In hepatocytes, glucose-1-phosphate is converted to glucose, by phosphoglucomutase and glucose-6phosphatase, which is then released into the blood. A summary of glycogenolysis is shown in Figure 1.10.13

Figure1.10 13.GlycogenDegradation:SummaryGlycogen phosphorylasecleavesthe α(1,4)linkages of glycogen to yield glucose-1-phosphateuntil it comeswithinfourglucose residues of abranchpoint.Debranchingenzymetransfers three of theseresidues to anearbynonreducing endand releasesthefourthresidue as freeglucose.Therepeated actions of bothenzymescanlead to thecompletedegradation of glycogen.

1.10.2 Lipid Catabolism

Snapshot

1. The fatty acids of triacylglycerols furnish a large fraction of the oxidative energy in animals. Dietary triacylglycerols are emulsified in the small intestine by bile salts, hydrolyzed by intestinal lipases, absorbed by intestinal epithelial cells, reconverted into triacylglycerols, then formed into chylomicrons by combination with specific apolipoproteins.

2. Once inside cells, fatty acids are activated at the outer mitochondrial membrane by conversion to fatty acyl CoA thioesters.

3. Fatty acyl CoA to be oxidized enters mitochondria in three steps, via the carnitine shuttle. In the first stage of oxidation, four reactions remove each acetyl-CoA unit from the carboxyl end of a saturated fatty acyl CoA:

4. In the second stage of fatty acid oxidation, the acetyl-CoA is oxidized to CO2 in the citric acid cycle. A large fraction of the theoretical yield of free energy from fatty acid oxidation is recovered as ATP by oxidative phosphorylation,the final

5. Malonyl-CoA, an early intermediate of fatty acid synthesis, inhibits carnitine acyltransferase I, preventing fatty acid entry

6. Genetic defects in the medium-chain acyl-CoA dehydrogenase result in serious human disease,as do mutations in other

7. Peroxisomes of plants and animals, and glyoxysomes of plants, carry out oxidation in four steps similar to those of the mitochondrial pathway in animals. The first oxidation step, however, transfers electrons directly to O2, generating H2O2. Peroxisomes of animal tissuesspecialize in the oxidation of very-long-chain fatty acids and branched fatty acids. In glyoxysomes, in germinating seeds, oxidation is one step in the conversion of stored lipids into a variety of intermediates

8. The reactions of oxidation, occurring in the endoplasmic reticulum, produce dicarboxylic fatty acyl intermediates, which

9. The ketone bodies are formed in the liver. The latter two compounds serve as fuel molecules in extrahepatic tissues, through oxidation to acetyl-CoA and entry into the citric acid cycle.

10. Overproduction of ketone bodies in uncontrolled diabetes or severely reduced calorie intake can lead to acidosis or

Storage Lipid/ Neutral Lipids (Triacylglycerols) undergo β oxidation to generate energy. More than half the energy of liver, heart, and resting skeletal muscle is obtained by oxidation of fatty acid

Properties of Triacylglycerol That Makes It An Efficient Storage Fuel

1. Highly Reduced structure:The long alkyl chains of their constituent fatty acids are essentially hydrocarbons, highly reduced structures with an energy of complete oxidation (~38 kJ/g) more than twice that for the same weight of carbohydrate or protein.

2. Unsolvated in nature: Since cellular triacylglycerols are insoluble in water they aggregate in lipid droplets, which do not raise the osmolarity of the cytosol, and they are unsolvated In contrast to storage polysaccharide which carries extra weight of water of solvation

3. Relative Chemical Inertness: because of their relative chemical inertness, triacylglycerols can be stored in large quantity in cells without the risk of undesired chemical reactions with other cellular constituents

Why Do Fatty Acids Undergo Β Oxidation ?



To overcome the relative stability of the C-C bonds in a fatty acid, the carboxyl group at C-1 is activated by attachment to coenzyme A, which allows stepwise oxidation of the fatty acyl group at the C-3, or β, position hence the name β oxidation.

Complete Oxidation Of Fatty Acid To Co2 And H2o Takes Place In Three Stages

The oxidation of long-chain fatty acids to two-carbon fragments, in the form of acetyl-CoA (β oxidation)

The oxidation of acetyl-CoA to CO2 in the citric acid cycle

And the transfer of electrons from reduced electron carriers to the mitochondrial respiratory chain Sources Of Fatty Acids

Fats consumed in the diet (Ingested Triacylglycerol)

Fats stored in cells as lipid droplets (Adipocytes)

Fats synthesized in one organ for export to another

in Liver from excess carbohydrates to fats for export to other tissues)

Steps For Fatty Acid Oxidation

Mobilization of fatty acid to the site where they required for energy generation

Activation of fatty acid in the cytosol

Transport of fatty acid across mitochondria via carnithine shuttle

Oxidation of fatty acid via

oxidation

1) Mobilization Of Ingested Triacyglyceerol To The Site Of Oxidation

Ingested triacylglycerols is converted from insoluble macroscopic fat particles to finely dispersed microscopic micelles.

solubilization is carried out by bile salts, such as taurocholic acid

Micelle formation enormously increases the fraction of lipid molecules accessible to the action of water-soluble lipases in the intestine.

Lipase action converts triacylglycerols to monoacylglycerols (monoglycerides) and diacylglycerols (diglycerides), free fatty acids, and glycerol.

The products of lipase action diffuse into the epithelial cells lining the intestinal surface (the intestinal mucosa) where they are reconverted to triacylglycerols and packaged with dietary cholesterol and specific proteins into lipoprotein aggregates called

dietarylipidsoccur

thesmallintestine,

Chylomicrons, which contain apolipoprotein C-II (apoC-II), move from the intestinal mucosa into the lymphatic system, and then enter the blood, which carries them to muscle and adipose tissue

In the capillaries of these tissues, the extracellular enzyme lipoprotein lipase, activated by apoC-II, hydrolyzes triacylglycerols to fatty acids and glycerol which are taken up either by muscle cells or adipocytes (Figure 1.10.14)

In muscle, the fatty acids are oxidized for energy however in the adipose tissue, they are reesterified for storage as triacylglycerols

The remnants of chylomicrons, depleted of most of their triacylglycerols but still containing cholesterol and apolipoproteins, travel in the blood to the liver, where they are taken up by endocytosis, mediated by receptors for their apolipoproteins.

These triacyglycerol could be either used for energy or converted to ketone bodies

When the diet contains more fatty acids than are needed immediately for fuel or as precursors, the liver converts them to triacylglycerols, which are packaged with specific apolipoproteins into VLDLs.

The VLDLs are transported in the blood to adipose tissues, where the triacylglycerols are removed and stored in lipid droplets within adipocytes.

Apolipoproteins

These

responsible for the transport of triacylglycerols, phospholipids, cholesterol, and cholesteryl esters between organs.

These are spherical aggregates with hydrophobic lipids at the core and hydrophilic protein side chains and lipid head groups at the surface.

The protein moieties of lipoproteins (ApoCII, ApoB48 and ApoCiii are recognized by receptors on cell surfaces which mediates lipase action

Various combinations of lipid and protein produce particles of different densities, ranging from chylomicrons and verylow- density lipoproteins (VLDL) to very-high-density

lipoproteins (VHDL)

Bile salts: the liver, stored in the gallbladder, and released into the small intestine after ingestion of a fatty meal.

Mobilization of Triacylglycerol Stored In Adipocytes

Neutral lipids are stored in adipocytes with a core of sterol esters and triacylglycerols surrounded by a monolayer of phospholipids

achylomicron. phospholipids,with head groupsfacingtheaqueousphase.Triacylglycerols morethan 80% themass.Severalapolipoproteinsthatprotrude as signals in theuptake and metabolism of chylomicroncontents. Thediameter of chylomicronsrangesfromabout 100 to 500nm

The surface of these droplets is coated with perilipins, a family of proteins that restrict access to lipid droplets, preventing untimely lipid mobilization

The hormones epinephrine and glucagon, secreted in response to low blood glucose levels, activate the enzyme adenylyl cyclase in the adipocyte plasma membrane (Figure 1.10.16)

This in turn produces the intracellular second messenger cyclic AMP.

Cyclic AMP activates protein kinase (PKA) which phosphorylates perilipin A

The phosphorylated perilipin causes hormone-sensitive lipase in the cytosol to move to the lipid droplet surface, where it can begin hydrolyzing triacylglycerols to free fatty acids and glycerol.

PKA also phosphorylates hormone-sensitive lipase, doubling or tripling its activity, but

Cells with defective perilipin genes have almost no response to increases in cAMP concentration

in adipocytes, the fatty acids thus released (free fatty acids, FFA) pass from the adipocyte into the blood, where they bind to the blood protein serum albumin (10 fatty acids per protein monomer)

Bound to this soluble protein,

are carried to tissues such as skeletal muscle, heart, and renal cortex.

In these target tissues, fatty acids dissociate from albumin and are moved by plasma membrane transporters into cells to serve as fuel.(Energy produced: 95% in Fatty Acid and 5% in Glycerol Backbone Produced via glycolysis)

Whenlowlevels of glucose in thebloodtriggertherelease of glucagon,1 thehormonebindsitsreceptor in theadipocytemembrane and thus2 stimulatesadenylylcyclase,viaaGprotein, to producecAMP.This activatesPKA,whichphosphorylates3thehormone-sensitivelipase and 4perilipinmolecules on thesurface of thelipiddroplet.Phosphorylation of perilipinpermitshormone-sensitivelipaseaccess to thesurface of thelipid droplet,where5 it hydrolyzestriacylglycerols to freefattyacids.6Fatty acidsleavetheadipocyte,bindserumalbumin in theblood, and are carried in theblood;theyarereleasedfromthealbumin and 7entera myocyteviaaspecificfattyacidtransporter.8 In themyocyte,fattyacids areoxidized to CO2, and theenergy of oxidation is conserved in ATP, whichfuelsmusclecontraction and otherenergy-requiringmetabolism in themyocyte.

This three-step process for transferring fatty acids into the mitochondrion

Activation Of Fatty Acid

enzyme acyl Co A Synthetase present

Energy bond)

to yield a fatty acyl CoA,

coenzyme

favorable by the hydrolysis of two high-energy bonds in

formation of a fatty acyl

reaction

reaction

the direction of fatty acyl CoA

immediately hydrolyzed by inorganic pyrophosphatase

reaction occurs

overall reaction

CoAs

Figure1. fattyacyl CoA thefattyacyl CoA derivativeoccurs

Fatty acyl CoA esters formed at the cytosolic side of the outer mitochondrial membrane can be transported into the mitochondrion and oxidized to produce ATP, or they can be used in the cytosol to synthesize membrane lipids

2) Transport Of Fatty Acid Across The Mitochondrion VI Carnithine Shuttle-

Fatty acid with 14 or more carbons, which constitute the majority of the FFA obtained in the diet cannot pass directly through the mitochondrial membranes

They must first undergo the three enzymatic reactions of the carnitine shuttle

Fatty acids destined for mitochondrial oxidation are transiently attached to the hydroxyl group of carnitine to form fatty acyl carnitine the second reaction of the shuttle.(ESTERIFICATION TO CARNITHINE)

o Enzyme: carnitine acyltransferase I in the outer membrane.

o Acyl Carnithine ester then passes into the intermembrane space (the space between the outer and inner membranes) through large pores (formed by the protein porin) in the outer membrane.

o The fatty acyl carnitine ester then enters the matrix by facilitated diffusion through the acylcarnitine/carnitine transporter of the inner mitochondrial membrane

the third and final step of the carnitine shuttle, the fatty acyl group is enzymatically transferred from carnitine to intramitochondrial coenzyme A by carnitine acyltransferase II ( present in the inner face of inner mitochondrial membrane) which in turn regenerates acyl Co-A and releases carnithine molecule

o Carnitine reenters the intermembrane space via the acyl-carnitine/carnitine transporter.

o The carnitine-mediated entry process is the ratelimiting step for oxidation of fatty acids in mitochondria .This is a regulation point.

o Once inside the mitochondrion, the fatty acyl CoA is acted upon by a set of enzymes in the matrix.

Oxidation of Fatty Acid

process in mitochondria(

processes

less-general modes

they occur

fatty-acylCoA producesadoublebondbetweenaandbcarbon,FAD is a succinatedehydrogenationsystem ETS)

thedouble bondof the thefumarasereaction

NADHdehydrogenase

eachpassthroughthis is removed in the thefattyacylchain in this palmitoyl-CoA.(b)Sixmorepasses acetyl-CoA,theseventh -carbonchain.Eight

-oxidation process in mitochondria

cycle of

oxidation

Acetyl Co-A molecule in turn

ATP

1.10.19)

other than mitochondria (Peroxisomes)

Acetyl Co-A (Figure 1.10.20)

ATP molecules

Acid (16 C) produces 8 Acetyl Co-A and 7 cycles of β oxidation will take place.

Oxidation Of Unsaturated Fatty Acids Requires Two Additional Reactions

Isomerase : The double bonds in monounsaturated fatty acid are in the cis configuration and cannot be acted upon by enoyl-CoA hydratase enzyme of β-oxidation ( enzyme catalyzing the addition of H2O to the trans double bond). Therefore Cis double bonds are converted to trans double bondΔ3,Δ2-enoyl-CoA isomerase which converts cis Δ3-enoyl-CoA to the trans Δ2-enoyl-CoA.

The no. of ATP produced during oxidation of unsaturated fatty acid is 1.5 molecules less than that produced during oxidation of saturated fatty acid

Reductase :

In case of polyunsaturated fatty acid For eg: Linoleic Acid undergoes three passes of -oxidation (Figure 1.10.21)

They are then been acted on isomerase which will convert cis- Δ3,cis Δ6 configuration trans- Δ2,cis Δ6 configuration and then undergoes one cycle of β-oxidation and the first step of reduction.

The Conjugated double bond formed is then acted by 2,4-dienoyl- CoA reductase, allowing reentry of this intermediate into the β oxidation pathway

Oxidation Of Odd Chain Fatty Acid

number of carbons are common in the lipids of many plants and some marine organisms. Cattle and other ruminant animals form large amounts of the three carbon propionate during fermentation of carbohydrates in the rumen. small quantities of propionate are added as a mold inhibitor to some breads and cereals, thus entering the human diet.

propionate is absorbed into the blood and oxidized by the liver and other tissues.

fatty acids

oxidized in the same pathway as the even-number acids, beginning at the carboxyl end of the chain. However, the substrate

for the last pass through the β oxidation sequence is a fatty acyl CoA with a five-carbon fatty acid.

When this is oxidized and cleaved, the products are acetyl-CoA and propionyl-CoA

The acetyl-CoA can be oxidized in the citric acid cycle, of course, but propionyl-CoA enters a

different pathway involving three enzymes finally getting converted to succinyl Co-A

1. propionyl-CoA carboxylase- Biotin (Co Factor)

2. methylmalonyl-CoA epimerase

3. methylmalonyl-CoA mutase- 5-deoxyadenosylcobalamin, or coenzyme B12 (Cofactor)

Regulation Of Fatty Acid Oxidation

When the diet provides a ready source of carbohydrate as fuel, β oxidation of fatty acids is unnecessary and is therefore downregulated (Figure 1.10.23)

Two enzymes are key to the coordination of fatty acid metabolism: acetyl-CoA carboxylase (ACC), the first enzyme in the synthesis of fatty acids, and carnitine acyl transferase I, which limits the transport of fatty acids into the mitochondrial matrix

for β oxidation.

Ingestion of a high-carbohydrate meal raises the blood glucose level and thus

triggers the release of insulin.

o Insulin-dependent protein phosphatase dephosphorylates ACC, activating it.

ACC catalyzes the formation of malonyl-CoA (the first intermediate of fatty acid synthesis)

malonyl-CoA inhibits carnitine acyltransferase

thereby preventing fatty acid entry into the mitochondrial matrix.

When blood glucose levels drop between meals,

Glucagon release activates cAMP-dependent protein kinase (PKA), which phosphorylates and inactivates ACC.

The concentration of malonyl-CoA falls, the inhibition of fatty acid entry into mitochondria is relieved and fatty acids enter the mitochondrial matrix and become the major fuel.

Because glucagon also triggers the mobilization of fatty acids in adipose tissue, a supply of fatty acids begins arriving in the blood.

Figure1.10 23.Coordinatedregulation

carbohydrate as fuel, β oxidation of fattyacids fattyacidmetabolism:acetyl-CoA carboxylase(ACC),thefirstenzyme whichlimitsthetransport of fatty acidsintothemitochondrialmatrixfor ahigh-carbohydratemealraisesthebloodglucoselevel and thus1 triggerstherelease .3ACCcatalyzestheformation of malonyl-CoA(thefirstintermediate therebypreventingfattyacidentry intothemitochondrialmatrix.Whenbloodglucoselevelsdropbetweenmeals,5glucagonreleaseactivatescAMP-dependentproteinkinase(PKA), which6phosphorylates fattyacidentryintomitochondria is relieved, and 7fattyacidsenterthemitochondrialmatrix fattyacids in adipose tissue,asupply

Perxisomal Oxidation

The mitochondrial matrix is the major site of fatty acid oxidation in animal cells (Figure 1.10.24).

In plant cells, the major site of

Perxisomal β oxidation of fatty acid is same as β oxidation taking place in mitochondria except:

The flavoprotein acyl-CoA oxidase that introduces the double bond passes electrons directly to

O2, producing H This strong and potentially damaging oxidant is immediately cleaved to H2O and O2 by catalase therefore the energy released in the first oxidative step of fatty acid breakdown is not conserved as ATP, but is dissipated as heat

The acetyl-CoA produced is converted via the peroxisomes and the glyoxylate cycle in plants are been used for synthesis of four-carbon precursors for gluconeogenesis

The peroxisomal system is much more active on very-longchain fatty acids such as hexacosanoic acid (26:0) and on branched-chain fatty acids such as phytanic acid and pristanic acid

These less-common fatty acids are obtained in the diet from dairy products, the fat of ruminant animals, meat, and fish.

Their catabolism in the peroxisome involves several auxiliary enzymes unique to this organelle. The inability to oxidize these compounds is responsible for several serious human Diseases

Zellweger syndrome are unable to make peroxisomes and therefore lack all the metabolism unique to that organelle

X-linked adrenoleukodystrophy (XALD), peroxisomes fail to long-chain fatty acids, apparently for lack for these fatty acids in the peroxisomal membrane.

ω oxidation

In some species, including vertebrates, that involves oxidation of the carbon from the carboxyl group( 1.10.25)

The enzymes unique to oxidation vertebrates) in the endoplasmic reticulum of liver and kidney, and the preferred substrates are fatty acids of 10 or 12 carbon atoms.

mitochondria and in glyoxysomes.Theperoxisomal/glyoxysomal tworespects:(1) in ,generating thesecondoxidativestep so reducing thecytosol,eventuallyentering peroxisomes and alsoexported;theacetatefromglyoxysomes germinatingseeds)serves as a mitochondria is

In mammals normally a minor pathway for fatty acid degradation, but when oxidation is defective (because of mutation or a carnitine deficiency, for example) it becomes more important.

α Oxidation

Phytanic acid has a methyl-substituted oxidation. (Figure 1.10.26)

The combined action of Phytanyl Co-A hydroxylase and Phytanyl Co-A lyase (TPP Cofactor) of the enzymes shown here removes the carboxyl carbon of phytanic acid, to produce pristanic acid, in which the β carbon is unsubstituted, allowing oxidation.

α oxidation of pristanic acid releases propionyl-CoA, not acetyl-CoA

Refsum’s disease, resulting from a genetic defect in phytanoyl-CoA hydroxylase, leads to very high blood levels of phytanic acid leading to neurological problems

Ketone Bodies

Most of the acetyl CoA produced by the oxidation of fatty acids in liver mitochondria undergoes further oxidation in the TCA cycle.

However, some of this acetyl CoA is converted to three important metabolites: acetone, acetoacetate, and b hydroxybutyrate. The process is known as ketogenesis, and these three metabolites are traditionally known as 1.10.27)

These three metabolites are synthesized primarily in the liver but are important sources of fuel and energy for tissues, including brain, heart, and skeletal muscle.

The brain, for example, normally uses glucose as its source of metabolic energy. However, during periods of starvation, ketone bodies may be the major energy source for the brain.

Acetoacetate and 3 hydroxybutyrate are the preferred and normal substrates for kidney cortex and for heart muscle.

The production and export of ketone bodies from the liver to extrahepatic tissues allow continued oxidation of fatty acids in the liver when acetyl-CoA is not being oxidized in the citric acid cycle.

theendoplasmic oxidationbeginswithoxidation of (omega)carbon. usuallyamedium-chainfattyacid;shownhere is generallynotthemajorroute

Ketone Body Synthesis

Ketone Bodies are synthesized in the mitochondria in the liver

The first reaction the condensation of two molecules of acetyl CoA to form acetoacetyl CoA is catalyzed by thiolase, which is also known as acetoacetyl-CoA thiolase or acetyl-CoA acetyltransferase. This is the same enzyme that carries out the thiolase reaction in β oxidation, but here it runs in reverse (Figure 1.10.28)

The second reaction adds another molecule of acetyl CoA to give β hydroxy β methylglutaryl CoA, commonly abbreviated HMG-CoA.

HMG-CoA is converted to acetoacetate and acetyl-CoA by the action of HMG-CoA lyase in a mixed aldol Claisen ester cleavage reaction.

Acetoacetate, is decarboxylated to acetone either spontaneously or by the action of acetoacetate decarboxylase

A membrane-bound enzyme, βhydroxybutyrate dehydrogenase, then can reduce acetoacetate to β-hydroxybutyrate.

Introduction of hydroxyl group: The oxygen for this group comes from molecular oxygen (O2) in a complex reaction that involves cytochrome P450 and the electron donor NADPH

Figure 1.10.27. Ketonebodies

Figure1.10 26.The α oxidation of abranched-chainfattyacid (phytanic acid) in peroxisomes.Phytanicacid has amethylsubstitutedcarbon and thereforecannotundergo oxidation.The combined action of theenzymesshownhereremovesthecarboxyl carbon of phytanicacid, to producepristanicacid, in whichthe carbon is unsubstituted,allowingoxidation.Noticethat oxidation of pristanic acidreleasespropionyl-CoA,notacetyl-CoA.

and β hydroxybutyrate are transported through the blood from liver to target organs and tissues, where they are converted to acetyl CoA which can then enter the Citric acid Cycle. Therefore Ketone Bodies could be used as fuel (Figure 1.10.29)

Alcohol dehydrogenase and alcohol dehydrogenase will act to convert the substarate to dicarboxylic acid.

Co A can added on either sides and the dicarboxylic acid can then undergo β oxidation

production and export of ketone bodies by the liver allow continued oxidation of fatty acids with only minimal oxidation of acetyl-CoA. Moreover it Provides Free Coenzyme A required for fatty acid Oxidation

bodies are easily transportable forms of fatty acids that move through the circulatory system without the need for complexation with serum albumin and other fatty acid binding proteins.

Normal Individuals, acetoacetate produced in small amounts which are easily decarboxylated to acetone.

with Diabetes produce large amount of acetoacetate and significant amount of acetone which gives characteristic odor to the breath, which is sometimes useful in diagnosing diabetes

Overproduction of Ketone bodies:

This happens during Starvation and during Diabetes

Starvation: gluconeogenesis depletes citric acid cycle intermediates, diverting acetyl-CoA to ketone body production

Untreated diabetes: When the insulin level is insufficient, extrahepatic tissues cannot take up glucose efficiently from the blood, either for fuel or for conversion to fat.

of malonyl- CoA fall due to which inhibition of carnitine acyltransferase I is relieved, and fatty acids enter mitochondria to be degraded to acetyl- CoA

This cannot pass through the citric acid cycle because cycle intermediates have been drawn off for use as substrates in gluconeogenesis.

The resulting accumulation of acetyl-CoA accelerates the formation of ketone bodies beyond the capacity of extrahepatic tissues to oxidize them.

The increased blood levels of acetoacetate and D-β hydroxybutyrate lower the blood pH, causing acidosis.

Increase in ketone bodies concentration in blood and urine leads to ketosis

Ketone bodies in the blood and urine of untreated diabetics

A blood concentration of 90 mg/100 mL (compared with a normal level of <3 mg/100 mL)

Urinary excretion of 5,000 mg/24 hr (compared with a normal rate of <125 mg/ 24 hr).

Amino Acid Metabolism

Snapshot

1. Amino acids are the precursors for numerous nitrogen-containing compounds such as heme, physiologically active amines, and glutathione.

2. Excess amino acids are converted to common metabolic intermediates for use as fuels.The first step in amino acid breakdown is removal of the amino group by transamination.

3. Transaminases require pyridoxal phosphate (PLP) and convert amino acids to their corresponding -keto acids. The amino group is transferred to-ketoglutarate to form glutamate, oxaloacetate to form aspartate, or pyruvate to form alanine.

4. Glutamate is subsequently oxidatively deaminated by glutamate dehydrogenase

5. (GDH) to form ammonia and regenerate ketoglutarate. Hyperinsulinism/hyperammonemia (HI/HA),a genetic disease,

6. In the urea cycle, amino groups from NH3 and aspartate combine with HCO3 to form urea. This pathway takes place

7. It begins with the ATP-dependentcondensation of NH3 and HCO3 by carbamoyl phosphate synthetase, an enzyme with a 96-Å-long tunnel connecting its three active sites through which its highly reactive intermediate products are

8. The resulting carbamoyl phosphate then combines with ornithine to yield citrulline, which combines with aspartate to

9. The arginine is then hydrolyzed to urea, which is excreted, and ornithine, which reenters the urea cycle. N-

10. The keto acid products of transamination reactions are degraded to citric acid cycle intermediates or their precursors.

11. The amino acids leucine and lysine are ketogenic in that they are converted only to the ketone body precursors acetyl-

12. The remaining amino acids are, at least in part, glucogenic in that they are converted to the glucose precursors 13. pyruvate, oxaloacetate, _-ketoglutarate, succinyl-CoA, or fumarate. Alanine, cysteine, glycine, serine, and threonineare

14. Serine hydroxymethyltransferase catalyzes the PLP-dependent CC bond cleavage of serine to form glycine. This -methylene-tetrahydrofolate, which the tetrahydrofolate (THF) obtains from the glycine cleavage system, a multienzyme system. Asparagine and aspartate are converted to

15. Ketoglutarate is a product of arginine, glutamate, glutamine, histidine, and proline degradation. Methionine, isoleucine,

16. S-adenosylmethionine (SAM), a sulfonium ion that acts as a methyl donor in many biosynthetic reactions. Hyperhomocysteinemia, a risk factor for cardiovascular disease, cognitive impairment and neural tube defects, is

17. Maple syrup urine disease (MSUD) is caused by an inherited defect in branched-chain amino acid degradation.

18.

19. Phenylalanine and tyrosine are degraded to fumarate and acetoacetate. Most individuals with the hereditary disease

The degradation of amino acids converts them to citric acid cycle intermediates or their precursors so that they can be metabolized to CO2 and H2O or used in gluconeogenesis (Figure 1.10.31).



Indeed, oxidative breakdown of amino acids typically accounts for 10 to 15% of the metabolic energy generated by animals.



The 20 “standard” amino acids (the amino acids of proteins) have widely differing carbon skeletons, so their conversions to citric acid cycle intermediates follow correspondingly diverse pathways. The fraction of metabolic energy obtained from amino acids, whether they are derived from dietary protein or from tissue protein, varies greatly with the type of organism and with metabolic conditions.

In animals, amino acids undergo oxidative degradation in three different metabolic circumstances:

1. During the normal synthesis and

2. When a diet is rich in protein and the

3. During starvation or in uncontrolled

aminoacidcatabolism in thecarbonskeletontake

Under all these metabolic conditions, amino acids lose their amino groups to form alpha-keto acids, the “carbon skeletons” of amino acids. The -keto acids undergo oxidation to CO2 and H2O or, often more importantly, provide three- and four-carbon units that can be converted by gluconeogenesis into glucose, the fuel for brain, skeletal muscle, and other tissues. (

One important feature distinguishes amino acid degradation from other catabolic processes described to this point: every amino acid contains an amino group, and the pathways for amino acid degradation therefore include a key step in which the -amino group is separated from the carbon skeleton and shunted into the pathways of amino group metabolism.

Amino acid metabolism takes place in two steps:

1. Amino acid deamination

2. Metabolism of the carbon skeleton

Amino acid deamination



The first reaction in the breakdown of an amino acid is almost always removal of its alpha-amino group with the object of excreting excess nitrogen and degrading the remaining carbon skeleton or converting (Figure 1.10.32)



Amino acids derived from dietary protein are the source of most amino groups. Most amino acids are metabolized in the liver.

Some of the ammonia generated in this process is recycled and used in a variety of biosynthetic pathways; the excess is either excreted directly or converted to urea or uric acid for excretion, depending on the organism.

Figure1. vertebrate liver.(b)Excretoryforms urea(most terrestrialvertebrates), and uric acidarehighlyoxidized;theorganismdiscardscarbononlyafterextractingmost itsavailableenergy of oxidation.

Glutamate and glutamine play especially critical roles in nitrogen metabolism, acting as a kind of general collection point for amino groups.

the cytosol of hepatocytes, amino groups from most amino acids are transferred to alpha ketoglutarate to form glutamate, which enters mitochondria and gives up its amino group to form NH4

ammonia generated in most other tissues is converted to the amide nitrogen of glutamine, which passes to the liver, then into liver mitochondria. Glutamine or glutamate or both are present in higher concentrations than other amino acids in most tissues.

skeletal muscle, excess amino groups are generally transferred to pyruvate to form alanine, another important molecule in the transport of amino groups to the liver.

Figure 1.10.32

TRANSAMINATION:

Most amino acids are deaminated by transamination, the transfer of their amino group to an alphaketo acid to yield the alpha-keto acid of the original amino acid and a new amino acid, in reactions catalyzed by aminotransferases (Figure 1.10.33).

Aminotransferase Reactions Occur in Two Stages

1. The amino group of an amino acid is transferred to the enzyme, producing the corresponding keto acid and the aminated enzyme.

2. The amino group is transferred to the keto acid acceptor (e.g., _-ketoglutarate), forming the amino

To carry the amino group, aminotransferases require participation of an aldehyde-containing coenzyme, pyridoxal-5

Esmond Snell, Alexander Braunstein, and David Metzler demonstrated that the aminotransferase reaction occurs via a Ping Pong Bi Bi mechanism whose two stages consist of three steps each

Stage I: Conversion of an Amino Acid to an alpha-Keto Acid

Step 1.The amino acids nucleophilic amino group

PLP Schiff base carbon atom in a transimination (trans-Schiffization) PLP Schiff base (aldimine), with concomitant release act as a general base at the active site.

Step 2 The amino acid PMP Schiff base by the active site Lys catalyzed removal of the amino acid _ hydrogen a resonance-stabilized carbanion intermediate. This resonance stabilization facilitates the cleavage of the Calpha-H bond.

Step 3.The α

Stage II: Conversion of an alpha-Keto Acid to an Amino Acid

To complete the aminotransferase’s catalytic cycle, the coenzyme must be converted from PMP back to the enzyme PLP Schiff base. This involves the same three steps as above, but in reverse order:

Step 3’ PMP reacts with an alpha-keto acid to form a Schiff base.

Step 2’ .The α -keto acid PMP Schiff base tautomerizes to form an amino acid PLP Schiff base.

Step 1’ The ε-amino group of the active site Lys residue attacks the amino acid PLP Schiff base in a transimination reaction to regenerate the active enzyme PLP Schiff base, with release of the newly formed amino acid. The reaction’s overall stoichiometry therefore is

The amino groups of most amino acids are consequently funneled into the formation of glutamate or aspartate, which are themselves interconverted by glutamate aspartate aminotransferase:

Figure1.10 33.Themechanism of PLP-dependentenzymecatalyzedtransamination.Thefirststage of the reaction, in whichthe-aminogroup ofan aminoacid is transferred to PLPyielding an -ketoacid and PMP,consists of threesteps:(1)transimination;(2)tautomerization, in whichtheLysreleasedduringthetransiminationreaction acts as ageneralacid basecatalyst; and (3)hydrolysis.Thesecondstage of thereaction, in whichtheaminogroup of PMP is transferred to adifferent-ketoacid to yielda new -aminoacid and PLP, is essentiallythereverse of the firststage:Steps3.,2., and 1.are,respectively,thereverse of Steps3,2, and 1.

The Glucose–Alanine Cycle Transports Nitrogen to the Liver

An important exception to the foregoing is a group of muscle aminotransferases that accept pyruvate as their alpha-keto acid substrate. The product amino acid, alanine, is released into the bloodstream and transported to the liver, where it undergoes transamination to yield pyruvate for use in gluconeogenesis. The resulting glucose is returned to the muscles, where it is glycolytically degraded to pyruvate. This is the glucose–alanine cycle (Figure 1.10.34).

The amino group ends up in either ammonium ion or aspartate for urea biosynthesis. Evidently, the glucose alanine cycle functions to transport nitrogen from muscle to liver (Figure 1.10.33).

Figure1.10 35.Theoxidativedeamination of glutamate by glutamatedehydrogenase.Thisreactioninvolvesthe intermediateformation of -iminoglutarate.

Oxidatative Deamination: Glutamate Dehydrogenase

Glutamate is oxidatively deaminated in the mitochondrial matrix by glutamate dehydrogenase (GDH).

Oxidation is thought to occur with transfer of a hydride ion from glutamate’s Calpha to NAD (P)+, thereby forming alphaiminoglutarate, which is hydrolyzed to alpha-ketoglutarate and ammonia.

GDH is allosterically inhibited by GTP, NADH, and nonpolar compounds such as palmitoyl-CoA and steroid hormones.

It is activated by ADP, NAD+, and leucine.

The ammonia generated by the process is transferred by glutamine.

Ammonia is quite toxic to animal tissues (we examine some possible reasons for this toxicity later), and the levels present in blood are regulated

In many tissues, including the brain, some processes such as nucleotide degradation generate free ammonia.

In most animals much of the free ammonia is converted to a nontoxic compound before export from the extrahepatic tissues into the blood and transport to the liver or kidneys. For this transport function, glutamate, critical to amino group metabolism, is supplanted by L-glutamine.

The free ammonia produced in tissues is combined with glutamate to yield glutamine by the action of glutamine synthetase.

This reaction requires ATP and occurs in two steps glutamate and ATP react to form ADP and aglutamyl phosphate intermediate, which then reacts with ammonia to produce glutamine and inorganic phosphate.

Figure1.10 36 Ammoniatransport in theform of glutamine. Excess ammonia in tissues is added to glutamate to form glutamine,aprocesscatalyzed by glutaminesynthetase.After transport in thebloodstream,theglutamineenterstheliver and NH4 is liberated in mitochondria by theenzymeglutaminase.

In most terrestrial animals, glutamine in excess of that required for biosynthesis is transported in the blood to the intestine, liver, and kidneys for processing. In these tissues, the amide nitrogen is released as ammonium ion in the mitochondria, where the enzyme glutaminase converts glutamine to glutamate and NH4+.

The NH4 from intestine and kidney is transported in the blood to the liver. In the liver, the ammonia from all sources is disposed of by urea synthesis.

deamination mechanism:

Two nonspecific amino acid oxidases, L-amino acid oxidase and D-amino acid oxidase, catalyze the oxidation of L- and

D-amino acids, utilizing FAD as their redox coenzyme [rather than NAD(P)+].The resulting FADH2 is reoxidized by O2.

D-Amino acid oxidase occurs mainly in kidney. Its function is an enigma since D-amino acids are associated mostly with bacterial cell walls. Urea Cycle And Nitrogen Excretion

If not reused for the synthesis of new amino acids or other nitrogenous products, amino groups are channeled into a single excretory end product.

Most aquatic species, such as the bony fishes, are excreting amino nitrogen as ammonia. The toxic ammonia is simply diluted in the surrounding water.

Terrestrial animals require pathways for nitrogen excretion that minimize toxicity and water loss. Most terrestrial animals are

Birds and reptiles are

In ureotelic organisms, the ammonia deposited in the mitochondria of hepatocytes is converted to urea in the This pathway was discovered in 1932 by Hans Krebs. Urea production occurs almost exclusively in the liver and is the fate of most of the ammonia channeled there.

Figure1.10 37.Nitrogen-acquiringreactions in thesynthesis of urea.Theureanitrogensareacquired in tworeactions,eachrequiringATP.

In thereactioncatalyzed by carbamoylphosphatesynthetase I, thefirstnitrogenentersfromammonia.Theterminalphosphategroups of twomolecules of ATPareused toformonemolecule of carbamoylphosphate. In otherwords,thisreaction has twoactivationsteps(1 and 3 ).(b) In thereactioncatalyzed by argininosuccinatesynthetase,thesecondnitrogenentersfromaspartate.Theureidooxygen of citrulline is activated by theaddition of AMP in step1;thissets up theaddition of aspartate in step2,withAMP(includingtheureidooxygen) as the leavinggroup.

The urea passes into the bloodstream and thus to the kidneys and is excreted into the urine.



The urea cycle begins inside liver mitochondria, butthree of the subsequent steps take place in the cytosol; the cycle thus spans two cellular compartments.



The mechanism of action is as discussed below:



A-Carbamoyl Phosphate Synthetase 1- It catalyzes the condensation and activation of NH3 and to form carbamoyl phosphate, the first of the cycle’s two nitrogen-containing substrates, with the concomitant hydrolysis of two ATPs.

The reaction catalyzed by CPS I involves three steps:

1. Activation of by ATP to form carboxyphosphate and ADP.

2. Nucleophilic attack of NH3 on carboxyphosphate, displacing the phosphate to form carbamate and Pi

3. Phosphorylation of carbamate by the second ATP to form carbamoyl phosphate and ADP. B-Ornithine Transcarboxylase: ornithine, yielding citrulline. The reaction occurs in the mitochondrion so that ornithine, which is produced in the cytosol, must enter the mitochondrion via a specific transport system.

The subsequent three steps take place in the cytosol

C-Argininosuccinate Synthetase-Urea’s second nitrogen atom is introduced in the urea cycle’s third reaction by the condensation of citrulline’s ureido group with an aspartate amino group by argininosuccinate The ureido oxygen atom is activated as a leaving group through formation of a citrullyl

D. Argininosuccinase- catalyzed elimination of arginine from the aspartate carbon skeleton forming fumarate. Arginine is urea’s immediate precursor. The fumarate produced in the argininosuccinase reaction reacts via the fumarase and malate dehydrogenase reactions to form oxaloacetate which is then used in gluconeogenesis

E.Arginase- catalyzed hydrolysis of arginine to yield urea and regenerate ornithine. Ornithine is then returned to the mitochondrion for another round of the cycle.



The urea cycle thereby converts two amino groups, one from NH3 and one from aspartate, and a carbon atom from to the relatively nontoxic excretion product urea at the cost of four “high energy” phosphate bonds (three ATP hydrolyzed to two ADP, two , and PP hydrolysis).

 This energetic cost, together with that of gluconeogenesis, is supplied by the oxidation of the acetylCoA formed by the breakdown of amino acid carbon skeletons.

Regulation of Urea cycle:

These changes in demand for urea cycle activity are met over the long term by regulation of the rates of synthesis of the four urea cycle enzymes and carbamoyl phosphate synthetase I in the liver (Figure 1.10.38)

Animals on protein-free diets produce lower levels of urea cycle enzymes. The first enzyme in the pathway, carbamoyl phosphate synthetase I, is allosterically activated by -acetylglutamate, which is synthesized from acetyl- CoA and glutamate by N-acetylglutamate synthase. N-acetylglutamate synthase activity in the liver has a purely regulatory function.

Understanding Biochem

-lactamases,whichprovidebacteriawithabulletproofvest.Abeta-lactamaseforms atemporarycovalentadductwiththecarboxylgroup of the opened beta-lactamring, which is immediatelyhydrolyzed,regeneratingactiveenzyme.Oneapproach to circumventingantibioticresistance of thistype is to synthesizepenicillinanalogs, suchas methicillin,thatare poor substratesfor betaactamases.Anotherapproach is to administeralongwithantibioticsabeta-lactamaseinhibitorsuch as clavulanate or sulbactam.

five enzymes involved are:

Carboxypeptidase

Ornithine transcarboxylase

Argininosuccinate synthetase

Argininosyccinase

Arginase

The yellow circle

Aspirinirreversiblyinactivatesthecyclooxygenaseactivity

acetylatinga

Figure1.10 39.Theureacycle.Itsfiveenzymesare(1)carbamoylphosphatesynthetase,(2)ornithinetranscarbamoylase,(3) argininosuccinatesynthetase,(4)argininosuccinase, and (5)arginase.Thereactionsoccur in part in themitochondrion and in part in thecytosolwithornithine and citrullinebeingtransportedacrossthemitochondrialmembrane by specifictransport systems(yellowcircles).One of theureaaminogroups(green)originates as the NH3product of theglutamatedehydrogenase reaction(top).Theotheraminogroup(red) is obtainedfromaspartatethroughthetransfer ofan aminoacid to oxaloacetatevia transamination(right).Thefumarateproduct of theargininosuccinasereaction is converted to oxaloacetateforentryinto gluconeogenesisviathesamereactionsthatoccur in thecitricacidcyclebuttakeplace in thecytosol(bottom).TheATPutilized in Reactions1 and 3 of thecyclecan be regenerated by oxidativephosphorylationfromtheNAD(P)Hproduced in theglutamate dehydrogenase(top)and malatedehydrogenase(bottom)reactions.

The degradation of amino acids converts them to citric acid cycle intermediates or their precursors so that they can be metabolized to CO2 and H2O or used in gluconeogenesis. Indeed, oxidative breakdown of amino acids typically accounts for 10 to 15% of the metabolic energy generated by animals. The overall process can be represented as shown in (Figure 1.10.40)

There are two main classes of amino acids:

1. Glucogenic amino acids: whose carbon skeletons are degraded to pyruvate, _-ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate and are therefore glucose precursors

2. Ketogenic amino acids, whose carbon skeletons are broken down to acetyl-CoA or acetoacetate and can thus be converted to ketone bodies or fatty acids

pathways and theimportance of agivenpathwaycanvarywiththeorganism and itsmetabolicconditions.Theglucogenic and ketogenicaminoacidsare alsodelineated in thefigure, by colorshading.Noticethatfive of theamino acidsarebothglucogenic and ketogenic.Theaminoacidsdegraded to pyruvatearealsopotentiallyketogenic.Onlytwoaminoacids,leucine and lysine,areexclusivelyketogenic.

Degradation of Alanine, cysteine, glycine, serine and threonine to pyruvate:

The carbon skeletons of six amino acids are converted in whole or in part to pyruvate. The pyruvate can then be converted to either acetyl-CoA (a ketone body precursor) or oxaloacetate (a precursor for gluconeogenesis).

The pathway is as follows:

1. Alanine is directly converted to pyruvate by the action of the enzyme alanine aminotransferase.

2. Serine is converted to pyruvate by serine dehydratase (Figure 1.10.41)

3. Glycine is converted to serine by the enzyme serine hydroxymethyltransferase, another PLPcontaining enzyme. This enzyme utilizes N5,N10-methylene-tetrahydrofolate (N5,N10-methyleneTHF) as a cofactor to provide the C1 unit necessary for this conversion

4. Threonine dehydrogenase- producing alpha-amino-alpha-ketobutyrate, which is converted to acetyl-CoA and glycine by alpha-aminoketobutyrate lyase.

Figure1.10 41.Thepathwaysconvertingalanine,cysteine,glycine,serine, and threonine to pyruvate.Theenzymesinvolvedare(1)alanine aminotransferase,(2)serinedehydratase,(3)glycinecleavagesystem,(4) and (5)serinehydroxymethyltransferase,(6)threoninedehydrogenase, and (7) α-amino-β-ketobutyratelyase.

Degradation of Asparagine and Aspartate to Oxoloacetate:

Asparagine is also converted to oxaloacetate in this manner after its hydrolysis to aspartate by Lasparaginase. The aspartate so formed can be directly converted to oxaloactate by transamination (Figure 1.10.42).

L-asparaginase is an effective chemotherapeutic agent in the treatment of cancers that must obtain asparagine from the blood, particularly acute lymphoblastic leukemia.The cancerous cells express particularly low levels of the enzyme asparagine synthetase and hence die without an external source of asparagine. However, L-asparaginase treatment may select for cells with increased levels of asparagine synthetase expression, and hence, in these cases, the surviving cancer cells are resistant to this treatment.

Degradation of Arginine, Glutamate, Glutamine, Histidine and proline to alpha-ketoglutarate:

1. Arginine is converted to ornithine by the enzyme arginase by utilizing a water molecule and the subsequent release of urea.

2. Ornithine is then converted to glutamate 5-semialdehyde by the enzyme ornithine deltaaminotransferase. This step involves the conversion of a molecule of alpha-ketoglutarate to glutamate

3. The cyclic structure of proline is opened by oxidation of the carbon most distant from the carboxyl group to create a Schiff base, then hydrolysis of the Schiff base to a linear semialdehyde, glutamate delta-semialdehyde. This reaction is catalyzed by proline oxidase.

4. The conversion of histidine to alpha-ketoglutarate is a more complex process and is done in the following steps (Figure 1.10.43)

5. The reaction is catalyzed by:

a) Histidine amino lyase converts histidine to Urocanate. In this process a molecule of ammonia is released.

b) Urocanate hydratase catalyses the conversion of urocanate to 4-imidazole-5-propionate utilizing a molecule of water.

c)Imidazolone propionate then converts it into N-formiminoglutamate utilizing a molecule of water

d) Glutamate formiminotransferase will convert this into glutamate.

Figure1.10

proline.Theseaminoacidsare converted 1histidineammonialyase,2 urocanatehydratase,3imidazolonepropionase,

Degradation of methionine, isoleucine, threonine and valine to Succinyl-CoA:

The methionine degradation is a more complex process and involves the following steps; (Figure 1.10.44)

o Methionine degradation begins with its reaction with ATP to form S-adenosylmethionine (SAM)

o Methylation reaction involving SAM yields S-adenosylhomocysteine in addition to methylated acceptor.

o The former product is hydrolyzed to homocysteine and adenosine.

The carbon skeletons of methionine, isoleucine, threonine, and valine are degraded by pathways that yield succinyl-CoA, an intermediate of the citric acid cycle.

Methionine donates its methyl group to one of several possible acceptors through S adenosylmethionine and three of its four remaining carbon atoms are converted to the propionate of propionyl-CoA, a precursor of succinyl-CoA

Isoleucine undergoes transamination, followed by oxidative decarboxylation of the resulting alphaketo acid. The remaining five-carbon skeleton is further oxidized to acetyl-CoA and propionyl-CoA.

Valine undergoes transamination and decarboxylation, then a series of oxidation reactions that convert the remaining four carbons to propionyl-CoA. Some parts of the valine and isoleucine degradative pathways closely parallel steps in fatty acid degradation.

Threonine is also converted in two steps to propionyl-CoA. This is the primary pathway for threonine degradation in humans. This reaction is catalyzed by the enzyme threonine dehydrogenase.

Understanding Biochem

In thehumanpopulationtherearethreecommonvariants, or alleles, of the gene encodingapolipoproteinE.Themostcommon,accountingforabout 78% of human apoE alleles, is APOE3;allelesAPOE4andAPOE2 account for 15% and 7%, respectively.TheAPOE4allele is particularlycommon in humanswith Alzheimer’s disease, and thelink is highlypredictive.IndividualswhoinheritAPOE4have an increasedrisk of late-onset Alzheimer’s disease.Thosewhoarehomozygousfor APOE4havea 16-foldincreasedrisk of developingthedisease;forthosewhodo, themean age of onset is just under 70 years. For peoplewhoinherittwocopies of APOE3, by contrast,themean age ofonset of Alzheimer’s disease exceeds90 years.

Degradation of leucine and lysine:

There are several pathways for lysine degradation, the one that proceeds via formation of the alphaketoglutarate predominates in mammalian liver. Oxidative decarboxylation of an alpha-keto acid by a multienzyme complex similar to pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. The enzymes involved in the pathway are as follows: (Figure 1.10.46)

1. Saccharophine dehydrogenase

2. Saccharophine dehydrogenase (NAD+, glutamate forming)

3. Aminoadipate semialdehyde dehydrogenase

4. Aminoadipate aminotraansferase (a PLP enzyme)

5. Alpha-keto acid dehydrogenase

6. Glutyrate-CoA dehydrogenase

7. Decarboxylase

8. Enoyl-CoA hydratase

9. Beta-hydoxyacyl=CoA dehydrogenase

10. HMG-CoA lyase

Figure1.10 46.Thepathway of lysinedegradation in mammalianliver.Theenzymesinvolvedare (1)saccharopinedehydrogenase(NADP,lysineforming), (2)saccharopinedehydrogenase(NAD, glutamateforming), (3)aminoadipatesemialdehydedehydrogenase,(4)Aminoadipate aminotransferase (a PLPenzyme), (5)-ketoaciddehydrogenase,(6)glutaryl-CoA dehydrogenase,(7)decarboxylase,(8)enoyl-CoAhydratase,(9)-hydroxyacylCoAdehydrogenase,(10)HMG-CoAsynthase, and (11)HMG-CoAlyase.

Breakdown of leucine:

branched chain amino acid is degraded by the following enzyme:

Branched chain amino-acid amino transferase. This catalyzes the formation of alpha-ketoisocapronic acid (Figure 1.10.47)

Figure1.

thebranched-chainaminoacids(leucine.Thefirstthree reactions of eachpathwayutilizethecommonenzymes(1)branched-chainaminoacid aminotransferase,(2)branched-chain-ketoaciddehydrogenase(BCKDH), and (3)acyl-CoA dehydrogenase.Isoleucinedegradationthencontinues(left)with (4)enoyl-CoAhydratase,(11)methylcrotonyl-CoAcarboxylase (a biotin-dependentenzyme),(12)-methylglutaconyl-CoA hydratase, and (13)HMG-CoAlyase to yieldacetyl-CoA and acetoacetate.

2. Branched chain alpha-keto acid dehydrogenase. It leads to the formation of isovaleryl-CoA

3. Acyl-CoA dehydrogenase leading to the formation of beta-methylcrotonyl-CoA

4. Beta-methylcrotonyl-CoA carboxylase leads to the formation of beta-methylglutaconyl-CoA

5. The next step is catalyzed by the enzyme beta-methylglutaconyl-CoA hydratase

6. Final step is catalyzed by HMG-CoA lyase to yield acetyl-CoA and acetoacetate.

dioxygenase,(2)formamidase,(3)kynurenine-3-monooxygenase,(4)kynureninase(PLPdependent),(5) 3-hydroxyanthranilate-3,4-dioxygenase,(6)aminocarboxymuconatesemialdehydedecarboxylase,(7) aminomuconatesemialdehydedehydrogenase,(8)hydratase,(9)dehydrogenase, )enzymes of ).2-Amino-3-carboxymuconate-6-semialdehyde, in NADPprecursor

enzymes involved are:

Tryptophan-2, 3-dioxygenase

Formamidase

Kynurenine-3-monooxygenase

Kynureninase (PLP dependent)

3-hydroxyanthranilate 3,4-dioxygenase

Amino carboxymuconate

Alpha-keto acid dehydrogenase

Glutyrate-CoA dehydrogenase

Decarboxylase

Enoyl-CoA hydratase

Beta-hydoxyacyl-CoA dehydrogenase

HMG-CoA lyase

16. HMG-CoA lyase

Degradation of phenylalanine and Tyrosine to Fumarate and Acetoacetate

Phenylalanine hydroxylase (also called phenylalanine- 4-monooxygenase) is one of a general class of enzyme called mixed-function oxidases. all of which catalyze simultaneous hydroxylation of a substrate by an oxygen atom of O2 and reduction of the other oxygen atom to H2O. Phenylalanine hydroxylase, requires the cofactor tetrahydrobiopterin, which carries electrons from NADH to O2 and becomes oxidized to dihydrobiopterin in the process. It is subsequently reduced by the enzyme dihydrobiopterin reductase in a reaction that requires NADH. (Figure 1.10.49).

Figure1.10 49 humanstheseaminoacidsarenormallyconverted to acetoacetyl-CoA

Genetic disorders related to amino acid metabolism: Alkaptonuria and Phenylketonuria Result from Defects in Phenylalanine Degradation-

Archibald Garrod realized in the early 1900s that human genetic diseases result from specific enzyme deficiencies. The first such disease to be recognized was alkaptonuria, which, Garrod observed, resulted in the excretion of large quantities of homogentisic acid. This condition results from deficiency of homogentisate dioxygenase. Alkaptonurics suffer no ill effects other than arthritis later in life (although their urine darkens alarmingly because of the rapid air oxidation of the homogentisate they excrete.

Severe mental retardation occurs within a few months of birth if the disease is not detected and treated immediately. Indeed, approx.1% of the patients in mental institutions were, at one time (before routine screening), phenylketonurics.



PKU is caused by the inability to hydroxylate phenylalanine and therefore results in increased blood levels of phenylalanine (hyperphenylalaninemia). The excess phenylalanine is transaminated to phenylpyruvate, by an otherwise minor pathway.



The “spillover” of phenylpyruvate (a phenylketone) into the urine was the first observation connected with the disease and gave the disease its name, although it has since been demonstrated that it is the high concentration of phenylalanine itself that gives rise to brain dysfunction.



Classic PKU results from a deficiency in phenylalanine hydroxylase (PAH).It was the first human inborn error of metabolism whose basic biochemical defect had been identified. Because all of the tyrosine breakdown enzymes are normal, treatment consists in providing the patient with a lowphenylalanine diet and monitoring the blood level of phenylalanine to ensure that it remains within normal limits for the first 5 to 10 years of life (the adverse effects of hyperphenylalaninemia seem to disappear after that age).



PAH deficiency also accounts for another common symptom of PKU: Its victims have lighter hair and skin color than their siblings. This is because tyrosine hydroxylation, the first reaction in the formation of the black skin pigment is inhibited by elevated phenylalanine levels.

 These result from deficiencies in the enzymes catalyzing the formation or regeneration of 5, 6, 7, 8tetrahydrobiopterin (BH4), the PAH cofactor. In such cases, patients must also be supplied with L3,4-dihydroxyphenylalanine

(L-DOPA) norepinephrine hydroxylase, the PAH homologs that produce these physiologically active amines, also require 5, 6, 7, 8-tetrahydrobiopterin

Defect in the lysine pathway:

The saccharopine pathway is thought to predominate in mammals because a genetic defect in the enzyme that catalyzes

The first step in the sequence results in

(elevated levels of lysine in the blood and This is yet another example of how the study of rare inherited disorders has helped to trace metabolic pathway.

Maple Syrup Urine Disease Results from a Defect in Branched-Chain Amino Acid Degradation

Branched-chain ketoisovalerate dehydrogenase),

catalyzes second step of branched-chain amino acid degradation. A genetic deficiency in BCKDH causes buildup of branched-chain alpha-keto acids imparts the urine with the characteristic odor of maple syrup. Unless promptly treated by a diet low in branched-chain amino acids (but not too low because they are essential amino acids) MSUD is rapidly fatal. MSUD is an autosomal recessive disorder that is caused by defects in any of four of the complex’s six subunits, E1, E1, E2, or E3 (E1 is an alpha2beta2 heterotetramer).

Hyperhomocysteinemia Is Associated with Disease

Imbalance between the rate of production of homocysteine through methylation reactions utilizing SAM and its rate of breakdown by either remethylation to form methionine or reaction with serine to form cystathionine in the cysteine biosynthesis pathway can result in an increase in the release of homocysteine to the extracellular medium and ultimately the plasma and urine. Moderately elevated concentrations of homocysteine in the plasma, hyperhomocysteinemia, for reasons that are poorly understood, are closely associated with cardiovascular disease, cognitive impairment, and neural

tube defects [the cause of a variety of severe birth defects including spina bifida (defects in the spinal column that often result in paralysis) and anencephaly (the invariably fatal failure of the brain to develop, which is the leading cause of infant death due to congenital anomalies)]. Hyperhomocysteinemia is readily controlled by ingesting the vitamin precursors of the coenzymes that participate in homocysteine breakdown, namely, B6 (pyridoxine, the PLP precursor; and folate

1.10.4 Nucleotide metabolism

Snapshot

1. The purine ring system is built up step-by-step beginning with 5-phosphoribosylamine. The amino acids glutamine, glycine, and aspartate furnish all the nitrogen atoms of purines. Two ring-closure steps form the purine nucleus.

2. Pyrimidines are synthesized from carbamoyl phosphate and aspartate, and ribose 5-phosphate is then attached to yield

3. Nucleoside monophosphates are converted to their triphosphates by enzymatic phosphorylation reactions. Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductase, an enzyme with novel mechanistic

4.

purines can be salvaged and rebuilt into nucleotides. Genetic deficiencies in certain salvage enzymes cause serious disorders suchas Lesch-Nyhan

5. Accumulation of uric acid crystals in the joints, possibly caused by another genetic deficiency,results in gout.

6. Enzymes of the nucleotide biosynthetic pathways are targets for an array of chemotherapeutic

7. agents used to treat cancer and other diseases.

Introduction

Uses of Nucleotides

1. Nucleotides are precursors of DNA and RNA and are essential carriers of chemical Energy a role primarily of ATP and to some extent GTP.

2. Components of the cofactors NAD, FAD, S-adenosylmethionine, and coenzyme A, well as of activated biosynthetic intermediates such as UDP-glucose and CDP-diacylglycerol.

3. cAMP and cGMP, are also cellular second messengers.

Nucleotide Biosynthesis (Figure 1.10.51)

1. ribonucleotides are formed first which is then utilized in the formation of corresponding deoxyribonucleotide.

2. Purine ring structure is built up of one or a few atoms at a time, attached to ribose throughout the process.

-5-phosphate

ATP

Salvagepathway

NucleotideBiosynthesis

pyrimidine

Phosphoribosyl

PRPP

DE NOVO PURINE SYNTHESIS

Denovo synthesis

and nucleosidesreleasedfromnucleic acidbreakdown Synthesis of nucleotidesbeginswith theirmetabolicprecursors:amino acids,ribose5-phosphate, CO2, and NH

and then converted to

acid

phosphate

is the

step of

of

purines.This informationwasobtainedfromisotopicexperimentswith supplied in the form of N

-formyltetrahydrofolate

of an amino group derived from aspartate

of inosinate

source

the high-energy phosphate

oxidation of inosinate at C-2, followed by addition of an amino group derived from glutamine

is formed by the NAD

Figure1.10 52 De novosynthesis of purinenucleotides:construction of thepurinering of inosinate (IMP).Eachaddition to thepurinering is shaded to matchFigure 22 32.Afterstep2,R symbolizesthe5-phospho-D-ribosylgroup on whichthepurinering is built.Formation of 5phosphoribosylamine(step1) is thefirstcommittedstep in purinesynthesis.Notethattheproduct of step9,AICAR, is theremnant of ATPreleasedduringhistidinebiosynthesis.Abbreviationsare givenformostintermediates to simplifythenaming of thepathwayenzymes.Step6a is the alternativepathfromAIR to CAIRoccurring in highereukaryotes.

Mechanisms of purine biosynthesis regulation.

of AMP

Allosteric regulation of enzyme glutamine transferase which transfer of an

to PRPP to form 5 phosphoribosylamine

Inhibits an enzyme IMP dehydrogenase which catalyze the conversion of inosinate

Mechanism 3

Enzyme is inhibited by Increased concentration of AMP and GMP

synthetase which catalyses conversion of inosinate to adenylosuccinate.

Does not affect the synthesis of GMP

Allosteric regulation of PRPP synthesis by the by inhibition of ribose phosphate pyrophosphokinase

Phosphorylation of AMP to ADP is promoted by adenylate kinase.

The ADP so formed is phosphorylated to ATP by the glycolytic enzymes or through oxidative phosphorylation.

ATP also brings about the formation of other nucleoside diphosphates by the action of a class of enzymes called nucleoside monophosphate kinases.

These enzymes are specific for a particular base but nonspecific for the sugar.

Nucleoside diphosphates are converted to triphosphates by the action of a ubiquitous enzyme, nucleoside diphosphate kinase and this enzyme sugar nor bases.

DE NOVO PYRIMIDINE SYNTHESIS

Common pyrimidine nucleotides are uridylate and cytidylate ( 1.10.53)

Thymidylate is derived from dCDP and dUMP

The pyrimidine ring is synthesized first and then attached to ribose 5phospahte.

Aspartate, carbamoyl phosphate and PRPP are the precursor of pyrimidine synthesis.

Carbamoyl phosphate reacts with aspartate to yield carbamoylaspartate in the first committed step of pyrimidine biosynthesis.

N-carbamoylaspartate is seen as an intermediate in urea cycle is made in mitochondria by carbamoyl phosphate synthetase I

N-carbamoylaspartate required for the pyrimidine biosynthesis is made in the cytosol by an enzyme called carbamoyl phosphate synthetase II in mammals while in bacteria single enzyme contribute carbamoyl phosphate to both arginine and pyrimidines.

Figure1.10 54 De novosynthesis of pyrimidinenucleotides: biosynthesis of UTP and CTPviaorotidylate.Thepyrimidine is constructedfromcarbamoylphosphate and aspartate.Theribose 5-phosphate is thenadded to thecompletedpyrimidinering by orotatephosphoribosyltransferase.Thefirststep in thispathway (notshownhere;see is thesynthesis of carbamoylphosphatefrom CO2and NH4,catalyzed in eukaryotes by carbamoylphosphate synthetase II

The first three enzymes in pyrimidine pathway carbamoyl phosphate synthetase II, aspartate transcarbamoylase, and dihydroorotase are part of a single trifunctional protein. This protein is called by and acronym CAD

regulation of pyrimidine synthesis :

o In bacteria, regulation occurs through aspartate carbamoyl transferase (ACTase) and is inhibited by CTP.

o The bacterial ACTase molecule consists of six catalytic subunits and six regulatory subunits.

o The bacterial ACTase exists in two conformations: active and inactive.

o When CTP is not bound the the regulatory subunits, the ACTase is active in maximum. Ribonucleotides Are The Precursors Of Deoxyribonucleotides

Deoxyribonucleotides, the building blocks of DNA, are derived from the corresponding ribonucleotides by direct reduction at the 2’-carbon atom of the D-ribose to form the 2’-deoxy derivative.

The enzyme catalyzing this reaction is called ribonucleotide reductase.

Glutaredoxin and thioredoxin transfer their reducing power to ribonucleotide reductase (Figure 1.10.55)

Figure1.10.55.Reduction of ribonucleotides to deoxyribonucleotides by ribonucleotidereductase.Electronsaretransmitted(bluearrows) to theenzyme fromNADPH by (a)glutaredoxin or (b)thioredoxin.Thesulfidegroups in glutaredoxinreductasearecontributed by twomolecules of boundglutathione (GSH;GSSGindicatesoxidizedglutathione).Notethatthioredoxinreductase is a flavoenzyme,withFAD as prostheticgroup.

Ribonucleotide reductase

The enzyme in E. coli and most eukaryotes is a dimer, with subunits designated R1 and R2.

The R1 subunit contains two kinds of regulatory sites (Figure 1.10.56)

Active site is at the interface of R1 and R2 subunit.

At each active site, R1 contributes two sulfhydryl groups required for activity and R2 contributes a stable tyrosyl radical.

The R2 subunit also has a binuclear iron (Fe3+) cofactor that helps generate and stabilize the tyrosyl radicals.

The tyrosyl radical is too far from the active site to interact directly with the site, but it generates another radical at the active site that functions in catalysis.

THYMIDYLATE IS DERIVED FROM dCDP AND dUMP

DNA contains thymine rather than uracil, and the pathway to thymine involves only deoxyribonucleotides

The immediate precursor of thymidylate (dTMP) is dUMP (Figure 1.10.57;Figure 1.10.58)

.Ribonucleotidereductase.(a)Subunitstructure. thetworegulatorysites.Eachactivesitecontains an active439,which

Figure 1.10.57. Biosynthesis of thymidylate(dTMP). The pathways are shown beginning with the reaction catalyzed by ribonucleotide reductase.

Purine catabolism

Adenosine deaminase deficiency causes severe immunodeficiency disease in which T-lymphocyte and B-lymphocyte do not develop properly.

o High concentration of dATPs causes deficiency of other dNTPs.

Figure1.10.59.Catabolism of purinenucleotides.Notethatprimatesexcretemuchmorenitrogen as ureaviatheureacycle (Chapter 18)than as uricacidfrompurinedegradation.Similarly,fishexcretemuchmorenitrogen as NH4than as ureaproduced by thepathwayshownhere.

Figure 1.10.60. Catabolism of a pyrimidine. Shown here is the pathway for thymine. The ethylmalonylsemialdehyde is further degraded to succinyl-CoA.

SALVAGE PATHWAY

Sl.No

Free adenine reacts with

AMP

the presence of enzyme adenosine phosphoribosyltransferase.

Guanine and hypoxanthine

product

adenine)

salvaged by hypoxanthine-guanine phosphoribosyl transferase.

syndrome

thymidylate synthase

nucleotide

the only cellular pathway for

synthesis.

Fluorouracil

converted to FdUMP by salvage pathway. FdUMP binds to enzyme and inhibit them

3. dihydrofolate reductase

conversion of dihydrofolate to tetrahydrofolate.

Methotrexate competitive inhibitor of enzyme.

enzyme

C

Allopurinol is used as drug to treat to gout, which inhibits xanthine oxidase which converts purine to uric acid.

Allopurinol is a substrate of xanthine oxidase, which converts allopurinol to oxypurinol (alloxanthine).

inactivates the reduced form of the enzyme by remaining tightly bound in its active site.

g lactone into

The pyrimidine moiety of riboflavin is biosynthetically related to guanine. Increased concentration of purine in media

plants, most fungi and bacteria, are phototrophic for Biotin. Others, including most vertebrates and some

In the form of a series of tetrahydrofolate (THF) compounds, folate derivatives are substrates in a number of single -phosphate) from dUMP

Vitamin A is required throughout life and participates in numerous cellular activities involved in reproduction, embryonic development, vision, growth, cellular differentiation and proliferation, tissue maintenance and lipid metabolism.

Vitamin D3 produced in the epidermis must be further metabolized to be active. The first step, 25-hydroxylation, takes

Ascorbate

Human cells cannot perform the crucial last step of vitamin C biosynthesis, the conversion of lgulono g lactone into ascorbic acid, which is catalysed by the enzyme gulonolactone oxidase.

Figure1.

itsanalogsAbbreviations: Ara/AraL,arabinose/arabinonolactone;Gal/GalA/GalL,galactose/galacturonicacid/galactonolactone;LGalDH,galactosedehydrogenase;L-GalLDH,galactonolactonedehydrogenase;D-GalUR,galacturonic acidreductase;Glc/GlcA/GlcL,glucose/glucuronicacid/gluconolactone;GulL,gulonolactone;L-GulO, gulonolactoneoxidase;GDP,guanosinediphosphate;Man,mannose;MeGalA,methylD-galacturonic acid;NDP,nucleosidediphosphate;UDP,uridinediphosphate.

VITAMIN B1

Thiamine diphosphate

Plants synthesize vitamin B1 (Thiamine diphosphate) in two mechanisms

1. donor) as seen in yeast and

5 aminoimidazole

the pyrimidine moiety of thiamine

amino 2 methyl 5 hydroxymethylpyrimidine

HMP P)

seen in bacteria.

Figure 1.10.62)

Figure1.10 62 of themajor thiaminediphosphate-incorporatingenzymes(B).TheArabidopsisgenesencodingtheenzymeswhichcatalyzethe biosyntheticreactionsshownarespecified.AIR,5-aminoimidazoleribonucleotide;HET- 4-methyl-5-(2hydroxyethyl)thiazolephosphate;HMP-P/HMP- 4-amino-2-methyl-5-hydroxymethylpyrimidine monophosphate/diphosphate;TA,thiamine;TMP,thiaminemonophosphate;TDP,thiaminediphosphate;PDC,pyruvate decarboxylase;PDH,pyruvatedehydrogenase;KGDH, -ketoglutaratedehydrogenase;BKDH,branched-chain α-ketoacid dehydrogenase;TK,transketolase;DXPS,1-deoxy-D-xylulose-5-phosphatesynthase.

VITAMIN B2

Riboflavin

(vitamin B2) is biosynthesized in plants and in many bacteria.

pyrimidine moiety of riboflavin

biosynthetically related to guanine.

concentration of purine in media increases the synthesis of riboflavin.

guanosine triphosphate (GTP) is the committed precursor of riboflavin supplying the pyrimidine ring and the nitrogen atoms of the pyrazine ring, as well as the ribityl side chain of the vitamin(Figure 1.10.63)

VITAMIN B3:

1. Quinolinate is the

2. In prokaryotes quinolinates are formed from dihydroxyacetone phosphate

3. In eukaryotes quinolinates are formed from tryptophan.

4. Humans lack an enzyme N-formylkynurenine formamidase (

VITAMIN B7

Higher plants, most fungi and bacteria, are phototrophic for Biotin. Others, including most vertebrates and some bacteria, rely on exogenous sources.

In mammals, Biotin is supplied by intestinal bacteria.

It consists of two fused rings: an Imidazol (Ureido) and a Sulf containing (Tetrahydrothiophene) ring; and the latter is extended via a Valeric acid side chain, which is attached in a configuration with respect to the Ureido ring.

Bacterial model for Biotin Bacillus subtilis), B. sphaericus(Bacillus sphaericus) and R. loti (Rhizobium loti).

The Biotin biosynthetic (bio) genes in the Gram Positive B. sphaericus are organized in two Operons located at different positions in the chromosome.

In B. sphaericus, Biotin is synthesized from Pimelic Acid involving the products of the bioW, X, F, A, D, and B genes of the bio Operon (Figure 1.10.65)

Folic acid

In the form of a series of tetrahydrofolate (THF) compounds, folate derivatives are substrates in a number of single-carbon-transfer reactions, and also are involved in the synthesis of dTMP (2′deoxythymidine-5′ phosphate) from dUMP (2′-deoxyuridine-5′ phosphate).

It is a substrate for an important reaction

involves vitamin B12 and it is necessary for the synthesis of DNA, and so required for all dividing cells.

The pathway leading to the formation of tetrahydrofolate (FH4) begins when folic acid (F) is reduced to dihydrofolate (DHF) (FH2), which is then reduced to THF. Dihydrofolate reductase catalyses the last step.

Vitamin B3 in the form of NADPH is a necessary cofactor for both steps of the synthesis. Thus, hydride molecules are transferred from NADPH to the C6 position of the pteridine ring to reduce

Methylene-THF (CH2FH4) is formed from THF by the addition of a methylene bridge from one of three carbon donors: formate, serine, or glycine. Methyl tetrahydrofolate (CH3-THF, or methylTHF) can be made from methylene-THF by reduction of the methylene group with NADPH.

Another form of THF, 10-formyl-THF, results from oxidation of methylene-THF or is formed from formate donating formyl group to THF. Also, histidine can donate a single carbon to THF to form

Vitamin B12 is the only acceptor of methyl-THF, and this reaction produces methyl-B12

producemethyl-vitaminB12

Thus, a deficiency in B12 can generate a large pool of methyl-THF that is unable to undergo reactions and will mimic folate deficiency.

The reactions that lead to the methyl-THF reservoir can be shown in chain form:

folate → dihydrofolate

VITAMIN B12

tetrahydrofolate

methylene THF → methyl-THF

Vitamin B12 normally plays a significant role in the metabolism of every cell of the body, especially affecting the DNA synthesis and regulation but also fatty acid synthesis and energy production.

However, many (though not all) of the effects of functions of B12 can be replaced by sufficient quantities of folic acid (vitamin B9), since B12 is used to regenerate folate in the body.

Most vitamin B12 deficiency symptoms are actually folate deficiency symptoms, since they include all the effects of pernicious anemia and megaloblastosis, which are due to poor synthesis of DNA when the body does not have a proper supply of folic acid for the production of thymine.

When sufficient folic acid is available, all known B12 related deficiency syndromes normalize, save those narrowly connected with the vitamin B12-dependent enzymes Methylmalonyl Coenzyme A mutase, and 5methyltetrahydrofolate-homocysteine methyltransferase (MTR), also known as methionine synthase; and the buildup of their respective substrates (methylmalonic acid, MMA) and homocysteine. Coenzyme B12's reactive C-Co bond participates in three main types of enzyme-catalyzed reactions.

Isomerases

substituents, an oxygen atom of an alcohol, or an amine. These use the adoB12

Methyltransferases

Methyl (-CH3) group transfers between two molecules. These use MeB12 (methylcobalamin) form

Dehalogenases

Reactions in which a halogen atom is removed from an organic molecule. Enzymes in this class

dependent enzyme families corresponding to the first two

MUT is an isomerase which uses the AdoB12form and reaction type 1to catalyze a carbon

MUT's reaction converts MMl-CoA to Su-CoA, an important step in the extraction of energy from proteins and fats (for more see MUT's reaction mechanism). This functionality is lost in vitamin B12 deficiency, and can be measured clinically as an increased methylmalonic

Unfortunately, an elevated MMA, though sensitive to B12 deficiency, is probably overly

98% of patients with B12 deficiency; however 20 25% of patients over the age of 70 have elevated levels of MMA, yet 25 33% of them do not have B12 deficiency.

o For this reason, assessment of MMA levels is not routinely recommended in the elderly. There is no "gold standard" test for B12 deficiency because as a B12 deficiency occurs, serum values may be maintained while tissue B12 stores become depleted.

o Therefore, serum B12 values above the cut-off point of deficiency do not necessarily indicate adequate B12 status.

o The MUT function cannot be affected by folate supplementation, which is necessary for myelin synthesis (see mechanism below) and certain other functions of the central nervous system.

o Other functions of B12 related to DNA synthesis related to MTR dysfunction (see below) can often be corrected with supplementation with the vitamin folic acid, but not the elevated levels of homocysteine, which is normally converted to methionine by MTR.

MTR, also known as methionine synthase, is a methyltransferase enzyme, which uses the MeB12 and reaction type 2 to catalyze the conversion of the amino acid homocysteine (Hcy) back into methionine (Met) (for more see MTR's reaction mechanism).

o This functionality is lost in vitamin B12 deficiency, and can be measured clinically as an increased homocysteine level in vitro. Increased homocysteine can also be caused by a folic acid deficiency, since B12 helps to regenerate the tetrahydrofolate (THF) active form of folic acid. Without B12, folate is trapped as 5-methyl-folate, from which THF cannot be recovered unless a MTR process reacts the 5-methyl-folate with homocysteine to produce methionine and THF, thus decreasing the need for fresh sources of THF from the diet.

o THF may be produced in the conversion of homocysteine to methionine, or may be obtained in the diet. It is converted by a non-B12-dependent process to 5,10-methyleneTHF, which is involved in the synthesis of thymine.

Reduced availability of 5,10-methylene-THF results in problems with DNA synthesis, and ultimately in ineffective production cells with rapid turnover, in particular blood cells, and

Figure 1.10.66. Metabolism of folic acid.The role of Vitamin B12

Thus the best-known "function" of B12 (that which is involved with DNA synthesis, celldivision, and anemia) is actually a facultative function which is mediated by B12conservation of an active form of folate which is needed for efficient DNA production. Other cobalamin-requiring methyltransferase enzymes are also known in bacteria, such

Absorption and distribution

The human physiology of vitamin B12 is complex, and therefore is prone to mishaps leading to vitamin B12 deficiency.

Protein-bound vitamin B12 must be released from the proteins by the action of digestive proteases in both the stomach and small intestine.

Gastric acid releases the vitamin from food particles; therefore antacid and acid-blocking medications (especially proton-pump inhibitors) may inhibit absorption of B12. In addition some elderly people produce less stomach acid as they age thereby increasing their probability of B12 deficiencies.

B12 taken in a low-solubility, non-chewable supplement pill form may bypass the mouth and stomach and not mix with gastric acids, but these are not necessary for the absorption of free B12 not bound to protein.

R-proteins (such as haptocorrins and cobalaphilin) are B12 binding proteins that are produced in the salivary glands. They must wait until B12 has been freed from proteins in food by pepsin in the



stomach. B12 then binds to the R-Proteins to avoid degradation of it in the acidic environment of the stomach.

This pattern of secretion of a binding protein secreted in a previous digestive step, is repeated once more before absorption. The next binding protein is intrinsic factor (IF), a protein synthesized by gastric parietal cells that is secreted in response to histamine, gastrin and pentagastrin, as well as the presence of food. In the duodenum, proteases digest R-proteins and release B12, which then binds to IF, to form a complex (IF/B12). B12 must be attached to IF for it to be absorbed, as receptors on the enterocytes in the terminal ileum of the small bowel only recognize the B12-IF complex; in addition, intrinsic factor protects the vitamin from catabolism by intestinal bacteria.



Absorption of food vitamin B12 thus requires an intact and functioning stomach, exocrine pancreas, intrinsic factor, and small bowel. Problems with any one of these organs makes a vitamin B12 deficiency possible. Individuals who lack intrinsic factor have a decreased ability to absorb B12. In pernicious anemia, there is a lack of IF due to autoimmune atrophic gastritis, in which antibodies form against parietal cells. Antibodies may alternately form against and bind to IF, inhibiting it from carrying out its B12 protective function. Due to the complexity of B12 absorption, geriatric patients, many of whom are hypoacidic due to reduced parietal cell function, have an increased risk of B12 deficiency. This results in 80 60% excretion in feces as seen in individuals with adequate IF.



Once the IF/B12 complex is recognized by specialized ileal receptors, it is transported into the portal circulation. The vitamin is then transferred to transcobalamin II (TC-II/B12), which serves as the plasma transporter. Hereditary defects in production of the transcobalamins and their receptors may produce functional deficiencies in B12 and infantile megaloblastic anemia, and abnormal B12 related biochemistry, even in some cases with normal blood B12 levels.[62] For the vitamin to serve inside cells, the TC-II/B12 complex must bind to a cell receptor, and be endocytosed. The transcobalaminII is degraded within a lysosome, and free B12 is finally released into the cytoplasm, where it may be transformed into the proper coenzyme, by certain cellular enzymes .



It's important to note that investigations into the intestinal absorption of B12 point out that the upper limit per single dose, under normal conditions, is about 1.5 µg: "Studies in normal persons indicated that about 1.5 µg is assimilated when a single dose varying from 5 to 50 µg is administered by mouth. In a similar study Swendseid et al. stated that the average maximum absorption was 1.6 µg [...]" [63]

 The total amount of vitamin B12 stored in body is about 2 5 mg in adults. Around 50% of this is stored in the liver.



Approximately 0.1% of this is lost per day by secretions into the gut, as not all these secretions are reabsorbed. Bile is the main form of B12 excretion; however, most of the B12 secreted in the bile is recycled via enterohepatic circulation. Excess B12 beyond the blood's binding capacity is typically excreted in urine.



Owing to the extremely efficient enterohepatic circulation of B12, the liver can store several years’ worth of vitamin B12; therefore, nutritional deficiency of this vitamin is rare. How fast B12 levels change depends on the balance between how much B12 is obtained from the diet, how much is secreted and how much is absorbed. B12 deficiency may arise in a year if initial stores are low and genetic factors unfavourable, or may not appear for decades. In infants, B12 deficiency can appear much more quickly.

FAT SOLUBLE VITAMINS

VITAMIN A

Retinol : Vitamin A has three active forms (retinal, retinol and retinoic acid) and a storage form (retinyl ester):

Retinyl ester ß à Retinol ß à Retinal à Retinoic acid

Circulating retinol is primarily bound to retinol-binding protein (RBP), and can enter and leave the liver several times per day in a process known as retinol recycling, which acts to relate the amount of retinol in circulation and protects cells from the damaging effects of free retinol or retinoic acid. Retinol bound to a cellular RBP (CRBP or CRBP-II) can be esterified by the enzyme lecithin:retinol acyltransferase (LCAT), the resulting retinyl ester being stored primarily in liver stellate cells. LCAT provides a readily retrievable storage form of vitamin A, as well as regulating its availability for other pathways.

Vitamin A is required throughout life and participates in numerous cellular activities involved in reproduction, embryonic development, vision, growth, cellular differentiation and proliferation, tissue maintenance and lipid metabolism. The three active forms of vitamin A each serve different overlapping functions. For instance, retinal is required for rhodopsin formation and vision, while retinoic acid is the principal hormonal metabolite required for proper growth and differentiation of

Vitamin A is required for the formation of the photoreceptor rhodopsin, which is a complex of retinal and the vision protein opsin, where retinal functions as the Rhodopsins are found in animals and green algae where they act as regulators of light-activated photochannels, and in archaea where they act as light-driven

In animals, the light-sensitive pigment rhodopsin occurs embedded in the

When light passes through the lens, it is sensed in the retina by both rod cells (black and white vision) and cone cells

In rod cells, the exposure of rhodopsin to light causes 11-cis-retinal to be released from opsin, resulting in a conformational change in the photoreceptor that activates theG-protein transducin. Transducin activation leads to the closure of the sodium channel in the membrane and the hyperpolarisation of the rod cell, which propagates a Rod cells are especially important for

Inadequate amounts of retinol can led to Night Blindness and corneal malformations, therefore eating carrots does let

Gene Expression and Vitamin A

Retinol and retinoic acid are important signalling molecules in vertebrates that act to alter

Several of these retinoidcontrolled genes are involved in growth and differentiation, such as those involved in the differentiation of the three germ layers, organogenesis and limb development during Retinoic acid exerts its effect through its binding to retinoic acid receptors (members of the steroid hormone superfamily of proteins), where the vitamin-receptor

Two families of receptors interact with vitamin A: the retinoic acid receptor (RAR) family that bind all-trans-retinoic acid (and 9-cis-retinoic acid), and the retinoic acid X receptor (RXR) family that bind only 9-cis retinoic acid. Together these receptors can regulate the rate of gene expression. Both vitamin A deficiency and excess can cause birth defects.

Immunity and Vitamin A

o Vitamin A is required for the normal functioning of the immune system. Retinol and its derivatives are required for the maintenance of the skin and mucosal cells that function as a barrier against infection, and are also required for the development of white blood cells that play a critical role in mounding an immune response. For example, the activation of T-cell lymphocytes requires the binding of the RAR receptor to retinoic acid. A deficiency in vitamin A can cause the mucosal membranes to atrophy, decreasing resistance to infection, and can increase the severity of infection. As such, vitamin A deficiency can be regarded as a nutritionally acquired immunodeficiency disease.

Cancer and Vitamin A

VITAMIN D

o Vitamin A intake has a complex relationship with cancer prevention: while small doses of vitamin A or beta-carotene appear to help prevent cancer, higher doses seem to have the reverse effect. The anti-cancer effects of beta-carotene appear to stem from its antioxidative ability to scavenge for reactive oxygen species, as well as through its conversion to vitamin A, which can improve immune function in addition to eliciting an anti-proliferative effect through the RAR and RXR receptors, thereby acting to block certain carcinogenic processes and inhibit tumour cell growth. However, an excessive intake of beta-carotene appears to have carcinogen effects, possibly through its promotion of the eccentric (or asymmetric) pathway of beta-carotene cleavage, which produces breakdown products that might lead to the destruction of retinoic acid through the activation of the P450 enzyme, which in turn could decrease retinoid signalling leading to enhanced cell proliferation. Therefore dosage seems to be an important factor in beta-carotene action.

Vitamin A is also involved in the production of red blood cells, which are derived from stem

In addition, vitamin A appears to facilitate the mobilisation of iron stores to developing red blood cells, where

Metabolism

Vitamin D3 produced in the epidermis must be further metabolized to be active. The first step, 25-hydroxylation, takes place primarily in the liver, although other tissues have this enzymatic activity as well. As will be discussed below, there are several hydroxylases. 25OHD is the major circulating form of vitamin D. However, in order for vitamin D metabolites to achieve maximum biologic activity they must be further hydroxylated in the 1α position by the enzyme CYP27B1; 1,25(OH)2D is the most potent metabolite of vitamin D and accounts for most of its biologic actions. The 1α hydroxylation occurs primarily in the kidney, although as for the 25-hydroxylase, other tissues have this enzyme. Vitamin D and its metabolites, 25OHD and 1,25(OH)2D, can also be hydroxylated in the 24 position. In the absence of 25-hydroxylation this may serve to activate the metabolite or analog as 1,25(OH)2D and 1,24(OH)2D have similar biologic potency. However, 24-hydroxylation of metabolites with an existing 25OH group reduces their activity and leads to further catabolism. The details of these reactions are described below.



Cutaneous production of vitamin D3. Although irradiation of 7-DHC was known to produce pre-D3 (which subsequently undergoes a temperature rearrangement of the triene structure to form D3), lumisterol, and tachysterol (figure 1), the physiologic regulation of this pathway was not well understood until the studies of Holick and his colleagues (9-11). They demonstrated that the formation of pre-D3 under the influence of solar or UV irradiation (maximal effective wavelength between 290310)is relatively rapid and reaches a maximum within hours. UV irradiation further converts pre-D3 to lumisterol and tachysterol. Both the degree of epidermal pigmentation and the intensity of exposure correlate with the time required to achieve this maximal concentration of pre-D3, but do not alter the maximal level achieved. Although pre-D3 levels reach a maximum level, the biologically inactive lumisterol continues to accumulate with continued UV exposure. Tachysterol is also formed, but like pre-D3, does not accumulate with extended UV exposure. The formation of lumisterol is reversible and can be converted back to pre-D3 as pre-D3 levels fall. At 0oC, no D3 is formed; however, at 37oC

pre-D3 is slowly converted to D3. Thus, short exposure to sunlight would be expected to lead to a prolonged production of D3 in the exposed skin because of the slow thermal conversion of pre-D3 to D3 and the conversion of lumisterol to pre-D3. Prolonged exposure to sunlight would not produce toxic amounts of D3 because of the photoconversion of pre-D3 to lumisterol and tachysterol as well as the photoconversion of D3 itself to suprasterols I and II and 5,6 transvitamin D3 .

 Melanin in the epidermis, by absorbing UV irradiation, can reduce the effectiveness of sunlight in producing D3 in the skin. This may be one important reason for the lower 25OHD levels (a well documented surrogate measure for vitamin D levels in the body) in Blacks and Hispanics living in temperate latitudes. Sunlight exposure increases melanin production, and so provides another mechanism by which excess D3 production can be prevented. The intensity of UV irradiation is also important for effective D3 production. The seasonal variation of 25OHD levels can be quite pronounced with higher levels during the summer months and lower levels during the winter. The extent of this seasonal variation depends on the latitude, and thus the intensity of the sunlight striking the exposed skin. In Edmonton, Canada (52oN) very little D3 is produced in exposed skin from midOctober to mid-April; Boston (42oN) has a somewhat longer period for effective D3 production; whereas in Los Angeles (34oN) and San Juan (18oN) the skin is able to produce D3 all year long . Peak D3 production occurs around noon, with a larger portion of the day being capable of producing D3 in the skin during the summer than other times of the year. Clothing and sunscreens effectively prevent D3 production in the covered areas. This is one likely explanation for the observation that the Bedouins in the Middle East, who totally cover their bodies with clothing, are more prone to develop rickets and osteomalacia than the Israeli Jews with comparable sunlight exposure.



Hepatic production of 25OHD. The next step in the bioactivation of D2 and D3, hydroxylation to 25OHD, takes place primarily in the liver although a number of other tissues express this enzymatic activity. 25OHD is the major circulating form of vitamin D and provides a clinically useful marker for vitamin D status. DeLuca and colleagues were the first to identify 25OHD and demonstrate its production in the liver over 30 years ago, but ambiguity remains as to the actual enzyme(s) responsible for this activity. 25-hydroxylase activity has been found in both the liver mitochondria and endoplasmic reticulum, and the enzymatic activities appear to differ suggesting different proteins. At this point most attention has been paid to the mitochondrial CYP27A1 and the microsomal CYP2R1. However, in mouse knockout studies and in human with mutations in these enzymes, only CYP2R1 loss is associated with changes in vitamin D metabolite production. These are mixed function oxidases, but differ in apparent Kms and substrate specificities.

 The mitochondrial 25-hydroxylase is now well accepted as CYP27A1, an enzyme first identified as catalyzing a critical step in the bile acid synthesis pathway. This is a high capacity, low affinity enzyme consistent with the observation that 25-hydroxylation is not generally rate limiting in vitamin D metabolism. Although initial studies suggested that the vitamin D3- -hydroxylase and cholestane triol 27-hydroxyase activities in liver mitochondria were due to distinct enzymes with differential regulation, the cloning of CYP27A1 and the demonstration that it contained both activities has put this issue to rest. CYP27A1 is widely distributed throughout different tissues with highest levels in liver and muscle, but also in kidney, intestine, lung, skin, and bone. Mutations in CYP27A1 lead to cerebrotendinous xanthomatosis , and is associated with abnormal vitamin D and/or calcium metabolism in some but not all of these patients . However, it is not clear that mutations in or even absence of CYP27A1 necessarily lead to cessation of vitamin D 25-hydroxylase activity despite leading to abnormalities in bile acid synthesis. CYP27A1 can hydroxylate vitamin D and related compounds atthe 24, 25, and 27 positions. However, D2 appears to be preferentially 24-hydroxylated, whereas D3 is preferentially 25-hydroxylated. The 1αOH derivatives of D are more rapidly hydroxylated than the parent compounds. These differences between D2 and D3 and their 1αOH derivatives may explain the differences in biologic activity between D2 and D3 or between 1αOHD2 and 1αOHD3.



The major microsomal 25-hydroxylase is CYP2R1, although other enzymes have been shown in in vitro studies to have 25-hydroxylase activity. This enzyme like that of CYP27A1 is widely distributed, although it is most abundantly expressed in liver, skin and testes. The skin expresses less and the

testes lack CYP27A1 expression. Unlike CYP27A1, CYP2R1 25-hydroxylates D2 and D3 equally (27). A patient with an inactivating mutation in CYP2R1 has been described with rickets and reduced 25OHD levels, reduced serum calcium and phosphate, but normal 1,25(OH)2D levels. The subject responded to D2 therapy. No other phenotype was reported in this subject, in particular no abnormalities in bile acid synthesis. Thus neither CYP27A1 nor CYP2R1 by themselves account for all 25-hydroxyase activity in the body, but each most likely contributes and together may account for most if not all of the 25-hydroxylase activity in humans.

 Studies of the regulation of 25-hydroxylation have not been completely consistent, most likely because of the initial failure to appreciate that at least two enzymatic activities were involved and because of species differences. In general, 25-hydroxylation in the liver is little affected by vitamin D status. However, CYP27A1 expression in the intestine and kidney is reduced by 1,25(OH)2D. Not surprisingly bile acids decrease CYP27A1 expression as does insulin through an unknown mechanism. Dexamethasone, on the other hand, increases CYP27A1 expression. The regulation of CYP2R1 has been less well studied. Whether any of these manipulations alters 25-hydroxylase activity in the liver remains unknown. Estrogen given to male rats increases 25-hydroxylase activity, whereas testosterone given to female rats has the opposite effect. However, evidence for such sex steroid hormone regulation of 25-hydroxylase activity in humans is lacking.



Renal production of 1,25(OH)2D. 1,25(OH)2D is the most potent metabolite of vitamin D, and mediates most of its hormonal actions especially those involving the vitamin D receptor (VDR), a transcription factor that will be discussed in the section on mechanism of action. 1,25(OH)2D is produced from 25OHD by the enzyme 25OHD-1αhydroxylase (CYP27B1). The recent cloning of CYP27B1 by four independent groups ended a long effort to determine the structure of this critical enzyme in vitamin D metabolism. Mutations in this gene are responsible for the rare autosomal disease of pseudovitamin D deficiency. An animal model in which the gene is knocked out by homologous recombination reproduces the clinical features of this disease including retarded growth, rickets, hypocalcemia, hyperparathyroidism, and undetectable 1,25(OH)2D.



CYP27B1 is a mitochondrial mixed function oxidase with significant homology to other mitochondrial steroid hydroxylases including CYP27A1 (39%), CYP24A1 (30%), CYPscc (32%), and CYP11β (33%) (35). However, within the heme-binding domain the homology is much greater with 73% and 65% sequence identity with CYP27A1 and CYP24A1. These mitochondrial P450 enzymes are located in the inner membrane of the mitochondrion, and serve as the terminal acceptor for electrons transferred from NADPH through ferrodoxin reductase and ferrodoxin. Expression of CYP27B1 is highest in epidermal keratinocytes (35) cells that previously had been shown to contain high levels of this enzymatic activity. However, the kidney also expresses this enzyme in the renal tubules as does the brain, placenta, testes, intestine, macrophages, lymph nodes, bone and cartilage.

more effectively than PTH and may be less inhibited by calcium, phosphate, and 1,25(OH)2D depending on the tissue. Administration of PTH in vivo or in vitro stimulates renal production of 1,25(OH)2D. This action of PTH can be mimicked by cAMP and forskolin indicating that at least part of the effect of PTH is mediated via its activation of adenylate cyclase. However, PTH activation of protein kinase C (PKC) also appears to be involved in that concentrations of PTH sufficient to stimulate PKC activation and 1,25(OH)2D production are below that required to increase cAMP levels . Furthermore, synthetic fragments of PTH lacking the ability to activate adenylate cyclase but which stimulate PKC activity were found to increase 1,25(OH)2D production. Direct activation of PKC with phorbol esters results in increased 1,25(OH)2D production. Although the promoter of CYP27B1contains several AP-1 (PKC activated) and cAMP response elements, it is not yet clear how PTH regulates CYP27B1 gene expression. Calcium modulates the ability of PTH to increase 1,25(OH)2D production. Calcium by itself can decrease CYP27B1activity and block the stimulation by PTH. Given in vivo, calcium can exert its effect in part by reducing PTH secretion, but this does not explain its direct actions in vitro or its effects in parathyroidectomized or PTH infused animals. Phosphate deprivation can stimulate CYP27B1 activity in vivo and in vitro. The in vivo actions of phosphate deprivation can be blocked by hypophysectomy and partially restored by growth hormone (GH) (64, 65) and insulin-like growth factor (IGF-I). However, like PTH, the exact mechanism by which

GH and/or

and TNF-

The principal regulators of CYP27B1 activity in the kidney are parathyroid hormone (PTH), FGF23, calcium, phosphate, and 1,25(OH)2D. Extrarenal production tends to be stimulated by cytokines such as IFN-,25(OH)2D administration leads to an apparent reduction in CYP27B1 activity . It was initially thought that this feedback inhibition was mediated at the level of gene expression. However, no vitamin D response element has been identified in the promoter of the 1α-hydroxylase gene. In keratinocytes, 1,25(OH)2D has little or no effect on CYP27B1 mRNA and protein levels when given in vitro. When 24-hydroxylase activity is blocked, 1,25(OH)2D administration fails to reduce the levels of 1,25(OH)2D produced. Thus the apparent feedback regulation of CYP27B1 activity by 1,25(OH)2D appears to be due to its stimulation of CYP24A1 and subsequent catabolism, not to a direct effect on CYP27B1 expression or activity at least in keratinocytes. However, in one renal cell line a chromatin remodeling complex (WINAC) has been described that mediates the ability of the vitamin D receptor to regulate CYP27B1 gene expression in a non classic manner enabling 1,25(OH)2D suppression of this gene; whether this mechanism is operative in other cells including normal kidney remains to be demonstrated. F-I mediates the effects of phosphate on CYP27B1 expression remains unclear. More recently FGF23 has been shown to inhibit CYP27B1 activity in vivo and in vitro. FGF23 has been implicated as at least one of the factors responsible for impaired phosphate reabsorption and 1,25(OH)2D production in conditions such as X-linked and autosomal dominant hypophosphatemic rickets and oncogenic osteomalacia .

 Renal production of 24,25(OH)2D. The kidney is also the major producer of a second important metabolite of 25OHD, namely 24,25(OH)2D, and the enzyme responsible is 25OHD-24 hydroxylase (CYP24A1). CYP24A1 and CYP27B1 are homologous enzymes that coexist in the mitochondria of tissues where both are found, such as the kidney tubule. However, they are located on different chromosomes (chromosome 20q13 and chromosome 12q14 for CYP24A1 and CYP27B1, respectively). They are likely to share the same ferrodoxin and ferrodoxin reductase components, although this has not been clearly established. While CYP27B1 activates the parent molecule, 25OHD, CYP24A1 initiates a series of catabolic steps that lead to its inactivation. However, in some tissues 24,25(OH)2D has been shown to have biologic effects different from 1,25(OH)2D as will be described subsequently. CYP24A1 requires a 25OH group, but can 24-hydroxylate both 25OHD and 1,25(OH)2D. The 24-hydroxylation is then followed by oxidation of 24OH to a 24-keto group, 23hydroxylation, cleavage between C23-24, and the eventual production of calcitroic acid, a metabolite with no biologic activity. CYP24A1 appears to catalyze all the steps in thiscatabolic pathway. Although CYP24A1 is highly expressed in the kidney tubule, its tissue distribution is quite broad including the intestine, osteoblasts, placenta, keratinocytes, and prostate. In general, CYP24A1 can be found wherever the VDR is found. The affinity for 1,25(OH)2D is higher than that for 25OHD, making this enzyme an efficient means for eliminating 1,25(OH)2D. Thus, CYP24A1 is likely to play the important role of protecting the body against excess 1,25(OH)2D. There are no known diseases due to CYP24A1 deficiency. However, an animal model in which CYP24A1 has been knocked out showed very high levels of 1,25(OH)2D and impaired mineralization of intramembranous bone. The skeletal abnormalities could be corrected by crossing this mouse to one lacking the VDR suggesting that excess 1,25(OH)2D (which acts through the VDR) rather than deficient 24,25(OH)2D (which does not) is to blame.

VITAMIN E

tocopherol

o Tocopherol contain a substituted aromatic ring and a long isoprenoid side chain.

o Because they are hydrophobic tocopherol associate with cell membranes, lipid droplets and lipoprotein.

o This vitamin is a natural antioxidant.

o The aromatic ring reacts with and destroys the most reactive forms of oxygen radicals and free radicals.

o Vitamin E deficiency is very rare; the principal symptom is fragile erythrocytes.

o Tyrosine is the precursor of tocopherol synthesis, which by converting to homogentisic acid is converted to tocopherol. (Figure 1.10.67)

Critical thinking Questions

1. When moleculesintomanysmall,simplemolecules.Underthesecircumstances,what on

2. In outsidethecycleprofoundlyaffectitsactivity.Whichones?

3. If youwerehandedasample wereatriacylglycerol

4. WhyareArachidonicacidandEPAareclassified

5. Fattyacidoxidationoccursmostlywithinmitochondria,butfattyacidsthemselvescannot easilycrossthemitochondrialmembrane. How do theypass?

1.11 ANSWER TO CRITICAL THINKING QUESTIONS

Structure of atoms, molecules and chemical bonds

1. What is the approximate percentage (in mass) of water in the human body? Is this percentage expected to be larger in the adult or in the old individual?

Ans) Approximately 65% of the human individual mass is water. The brain, for example, has around 90% of water in mass, the muscles, 85%, and the bones have between 25% and 40% of water. Younger adult individuals have proportionally more water in mass than older individuals.

2. Can the heat capacity of water be considered small or large? What is the biological significance of that characteristic?

Ans) Water has a specific heat of 1 cal/g.oC which means that 1 oC per gram is changed in its temperature with the addition or subtraction of 1 cal of energy. This is a very elevated value (for example, the specific heat of ethanol is 0.58 cal/g °C, and mercury, a metal, has a specific heat of 0.033 cal/g. °C) making water an excellent thermal protector against variations of temperature. Even if sudden external temperature changes occur, the internal biological conditions are kept stable in organisms which contain enough water.

3. Which of the three species CO3 2-, CO2 and CO has the shortest C-O bond length? Explain the reason for your answer.

Ans) CO has the shortest bond because there is a triple bond, or because the bond order is 3, meaning it has the greatest number of shared electrons between the carbon and the oxygen atom.

4. Ammonia, NH

Ans) Ammonia has hydrogen-bonding intermolecular forces, whereas phosphine has dipole- dipole and/or dispersion intermolecular forces. Water also has hydrogen-bonding intermolecular attractive forces. Ammonia is more soluble in water than phosphine because ammonia molecules can hydrogen-bond with water molecules, whereas phosphine molecules cannot hydrogen-bond with water molecules.

5. Which of the following describes the changes in forces of attraction that occur as H O changes phase from a liquid to a vapor? A. H-O bonds break B. Hydrogen bonds break C. Ionic bonds break D. Covalent bonds become more effective.

Ans) B

Composition, structure and function of biomolecules (carbohydrates, lipids, proteins, nucleic acids and vitamins)

1. Why is glycine a highly conserved amino acid residue in the evolution of proteins?

Ans. Glycine has the smallest side chain of any amino acid. Its smallness is critical in allowing polypeptide chains to make tight turns or to approach one another closely.

2. The gene encoding a protein with a single disulphide bond undergoes a mutation that changes a serine residue into a cysteine residue. Propose a direct method to find out whether the disulphide pairing in this mutant is the same as in the original protein.

Ans. The positions of the disulphide bond can be determined by diagonal electrophoresis. The disulphide pairing is unaltered by the mutation if teh off-diagonal peptides formed fromt eh native and mutant proteins are the same.

3. The atmosphere of the primitive earth before the emergence of life contained N2, NH3, H2, HCN, CO and H2O. Which of these compounds is the most likely precursor of most of the atoms in adenine? Why?

Ans. HCN. Adenine can be viewed as a pentamer of HCN.

4. Are organic solvents like benzene and ether polar or non-polar substances?

Ans) Benzene and the ethers are molecules without electrically charged portions and thus they are non-polar substances.

5. How to distinguish between glucose and sucrose?

Ans) Glucose is reducing sugar, it reacts with tollens reagent to form silver mirror, while sucrose is a non-reducing sugar. It does not react.

Stabilizing interactions (Van der Waals, electrostatic, hydrogen bonding, hydrophobic interaction, etc.)

1. Why does Poly-L-leucine, in an organic solvent like dioxane, is alpha-helical whereas poly-L-isoleucine is not?

Ans. The methyl group attached to the beta carbon of isoleucine sterically interferes with alpha helix formation. In leucine this methyl group is attached to the gamma carbon atom, which is farther from the main chain and hence does not interfere.

2. What type of non-covalent interactions holds together Graphite?

Ans) van der Waals Interactions

3. A mutation that changes Ala to Val in the interior of a protein leads to loss of activity. A 2nd mutation in different position changes Isoleucine to glycine. How does this restore the activity?

Ans) The first mutation destroys activity because valine occupies more space than alanine does, so protein must take a different shape, assuming this residue lies in the closely packed interior. The second mutation restores activity because of compensatory reduction in volume; glycine is smaller than isoleucine.

4. Why is it not correct to assert that DNA self-replicates?

Ans) DNA is not completely autonomous in its duplication process because the replication does not occur without enzymatic activity. So it is not entirely correct to assert that DNA self-replicates.

5. Do the phosphate and the pentose groups give homogeneity or heterogeneity to the nucleic acid chains? What about the nitrogen-containing groups?

Ans) The phosphate and the pentose groups are the same in every nucleotide that forms the nucleic acid and so they give homogeneity to the molecule. The nitrogen-containing bases however can vary among adenine, thymine, cytosine, guanine (in DNA) and uracil (in RNA). These variations provide the heterogeneity of the nucleic acid molecule.

Principles of biophysical chemistry (pH, buffer, reaction kinetics, thermodynamics, colligative properties)

1. What is the 'pH' of pure water and that of rain water? Explain the difference.

The pH of pure water is seven. Rain water is slightly acidic because as rain drop fall, the carbon dioxide in the air dissolves with drops to form very weak carbonic acid. Accordingly, rain water has a pH that is slightly below 7.

2. What is the pH of solution 'A' which liberates CO2 gas with a carbonate salt? Give the reason?

The pH of solution 'A' is lesser than 7. Carbonates salts react with acids (A) to liberate CO2 gas.

3. What is a universal indicator? What is its advantage?

A universal indicator is a mixed indicator of organic chemicals which not only shows whether the given solution is acidic or basic, but also shows the approximate pH values by giving a wide particular colour for a specific value of pH.

4. Why can't hydrogen ions exist by themselves?

Ans) It is possible to have isolated H+ ions in the gas phase. But if a free hydrogen ion encounters a water molecule, it attacks the unshared electron pairs on the oxygen in the water molecule and forms a hydronium ion, H . The chemical bond that forms between the water and hydrogen ion is covalent and very strong. In an aqueous solution, essentially all of the H+ exists as H

5. Is a negative pH possible?

Ans) It's possible. If the molarity of hydrogen ions is greater than 1, you'll have a negative value of pH. For example, you might expect a 12 M HCl solution to have a pH of -log(12) = -1.08. There are some complications in high molarity acid solutions that make pH calculations from acid molarity inaccurate and difficult to verify experimentally.

Bioenergetics, glycolysis, oxidative phosphorylation, coupled reaction, group transfer, biological energy transducers

1. Suppose that you wound a spring tightly, clamped it so that it could not unwind, and then placed the spring in acid, dissolving it without unwinding. What would happen to the energy stored in the spring?

Ans) The solution becomes Warm.

2. Suppose that you determined G for a reaction at room temperature and then carried out the same reaction at a higher temperature. At higher temperatures, what will the change in G for the reaction as compared to that at lower temperatures?

Ans) A larger negative number than at room temperature.

3. The first step in glycolysis is phosphorylation of glucose to form glucose-6-phosphate. What is the importance of this step?

Ans) To make glucose more polar, locking it within the cell.

4. If we can convert glucose to pyruvic acid and to other metabolites, we should be able to simply reverse glycolysis and form new glucose from pyruvic acid. What prevents this?

Ans) The free energy changes for some of the reactions that lead from glucose to pyruvate are too large and negative for easy reversal.

5. Oligomycin Inhibits synthesis of ATP during oxidative phosphorylation. What is the mechanism of inhibition?

Ans) Oligomycin inhibits mitochondrial ATPase and thus prevents phosphorylation of ADP to ATP. It prevents utilization of energy derived from electron transport for the synthesis of ATP. Oligomycin has no effect on coupling but blocks mitochondrial phosphorylation so that both oxidation and phosphorylation cease in its presence.

Principles of catalysis, enzymes and enzyme kinetics, enzyme regulation, mechanism of enzyme catalysis, isozymes

1. Suppose a mutant enzyme binds a substrate 100-fold as tightly as does the native enzyme. What is the effect of this mutation on the catalytic rate if the binding of the transition state is still unaffected?

Ans. The mutation slows the reaction by a factor of 100 because the activation free energy is increased by +2.72 kcal/mol (2.303RTlog100). Strong binding of the substrate relative to the transition state slows catalysis.

2. What is a simple means of determining whether a recently discovered proteolytic enzyme is a thiol protease?

Ans. The essential sulfhydryl groups in its active site should be highly susceptible to chemical modification. The enzyme should be inactivated by reacting it with iodoacetamide, N-ethylmaleimide or any other sulfhydryl-specific reagent. Bound sulfhydryl is likely to protect the key sulfhydryl from alkylation.

3. The HIV 1 protease, like other retroviral proteases, is a dimer of identical subunits rather than a single chain twice as large. What is the selective advantage to the virus of a dimeric arrangement?

Ans. Less RNA is needed to encode the protease. Small genome is more readily replicated and packaged than a large one. Also, the protease does not become active until a critical concentration is attained. Premature excision of the viral subunits from the polyprotein is thereby prevented.

4. You have isolated a dimeric enzyme that contains 2 identical active sites. The binding of substrate to one active site decreases the substrate affinity of the other active site. Which allosteric model best accounts for this negative cooperativity?

Ans. The sequential model can account for the negative cooperativity, whereas the concerted model cannot. Homotropic allosteric interactions should be cooperative if concerted model holds.

5. Antithrombin III forms an irreversible complex with thrombin but not with prothrombin. What is the most likely reason for this difference in reactivity?

Ans. Antithrombin III is a very slowly hydrolyzed substrate of thrombin. Hence its interaction with thrombin requires a fully formed active site on the enzyme.

Conformation of proteins (Ramachandran plot, secondary structure, domains, motif and folds)

1. How can curls be induced in hair?

Ans. Disulphide bonds in hair are broken by adding a thiol and applying gentle heat. The hair is curled and an oxidising agent is added to reform the disulphide bonds and stabilize the desired shape.

2. Suppose that a protease is synthesized by the solid-phase method from D rather than L amino acids. How would the sedimentation, electrophoretic and circular dichroism properties of this enzyme compare with those of the native form?

Ans. The sedimentation and electrophoretic properties of the L-enzyme and the mirror image D form would be the same. The circular dichroism spectra would have the same magnitude but be of opposite sign because the two structures have opposite screw sense.

3. Proteins that span biological membranes often contain alpha helices. Why is an alpha-helix particularly suited to exist in a hydrophobic environment?

Ans) The amino acids in the alpha helix would be hydrophobic in nature. All the amide hydrogen atoms and carbonyl oxygen atoms of the peptide backbone take part in intrachain H bonds, thus stabilizing these polar atoms in a hydrophobic environment.

4. What is the essential condition for a protein to be identical to another protein?

Ans) For a protein to be identical to another protein it is necessary for the sequence of amino acids that form them to be identical.

5. In sickle cell anemia, are all of the structural levels of the protein modified?

Ans) In sickle cell disease there is a change in the primary protein structure of one of the polypeptide chains that form hemoglobin: the amino acid glutamic acid is substituted by the amino acid valine in the chain. The spatial conformation of the molecule in addition is also affected and modified by this primary and the modification also creates a different (sickle) shape to the red blood cells. Modified, sickled, red blood cells sometimes aggregate and obstruct the peripheral circulation causing tissue hypoxia and the pain crisis typical of sickle cell anemia.

Conformation of nucleic acids (helix (A, B, Z), t-RNA, micro-RNA)

1. How does cordycepin (3’

Ans. Cordycepin terminates RNA synthesis. An RNA chain containing cordycepin lacks a 3’ OH group.

2. A negatively supercoiled DNA molecule undergoes a B to Z transition over a segment of 360 base pairs. What is the effect on the writhing (supercoiling)?

Ans) +66

The twist changes from that in B-form (TB) to that in Z DNA (TZ):

TB = 360/ +10 = +36 and TZ = 360/-12 = -

ΔT = TZ - TB = -

ΔT = TZ T

ΔL = 0

ΔW = -ΔT = -(-66) = +66

3. Why is HAP column used to distinguish single stranded and double stranded DNA?

Ans) HAP (hydroxyapatite) column. Duplex nucleic acids will bind to HAP at room temperature, whereas single-stranded nucleic acids will elute. The duplex fraction can subsequently be retrieved from the column by heating it, melting the nucleic acid and now collecting it as it elutes.

4. To precipitate DNA, an alcohol like ethanol or propanol is added to an aqueous DNA solution. Why should Na+ or NH4+ also be added?

Ans) a counter ion (Na+ or NH4+) must be present in order for the negatively-charged DNA to form a salt and precipitate.

5. A 41.5 nm-long duplex DNA molecule in the B-conformation adopts the A conformation upon dehydration How long is it now? What is its approximate number of base pairs?

Ans) 27.3 nm and 122 bps

For B-DNA, 41.5 nm has 41.5/0.34 = 122 bps that make 122/10 = 12.2 turns. If converted to A-DNA, these 122 base pairs will now make: 122/11 = 11.1 turns with an overall length of 122 * 0.224 = 27.3nm

Stability of proteins and nucleic acids

1. Suppose you want to radioactively label DNA but not RNA in dividing and growing bacterial cells. Which radioactive molecule would you add to the culture medium?

Ans) Tritiated thymine or tritiated thymidine

2. RNA is readily hydrolyzed by alkali whereas DNA is not. Why?

Ans) The 2’ OH group in RNA acts as an intramolecular nucleophile. In the alkaline hydrolysis of RNA, it forms a 2’ 3’ cyclic intermediate.

3. The amino acid coded by codon GGG could not be deciphered in the same way as UUU, CCC or AAA. Why is polyG an ineffective template?

Ans) PolyG forms a triple helical structure. Only single stranded RNA can serve as a template for protein synthesis.

4. Griffith used heat-killed S. Pneumococci to transform R mutants. Studies years later showed that dsDNA is needed for efficient transformation and that high temperature melt the DNA double helix. Why were Griffith’s experiments nevertheless successful?

Ans. The DNA renatured when the heat killed pneumococci were cooled before they were injected into mice.

5. In designing of primers, the Tm of each primer should approximately be the same. What is the basis of this requirement?

Ans) If the Tm of the primers are too different, the extent of hybridization with the target DNA will differ during the annealing phase, which would result in differential replication of the strands.

Metabolism of carbohydrates, lipids, amino acids nucleotides and vitamins

1. When we digest food and use it to produce energy, we convert a few large, complex molecules into many small, simple molecules. Under these circumstances, what is the effect on entropy?

Ans) Entropy increases.

2. In addition to the regulators of enzyme activity within the citric acid cycle, two enzymes outside the cycle profoundly affect its activity. Which ones?

Ans) Pyruvate carboxylase and pyruvate dehydrogenase.

3. If you were handed a sample of a white, greasy substance and asked to determine whether it were a triacylglycerol or a fatty acid, how would you do it?

Ans) By mixing it with base to see whether it dissolved. Fatty acids readily react with bases to form soap solutions.

4. Why are Arachidonic acid and EPA are classified as essential?

Ans) They are needed for synthesis of eicosanoids.

5. Fatty acid oxidation occurs mostly within mitochondria, but fatty acids themselves cannot easily cross the mitochondrial membrane. How

Ans) As esters of carnitine.

Test Yourself Test Yourself

1. The peptide bonds after positively charged amino acids are cleaved by?

a. Cyanogen bromide

b. Chymotrypsin

c. Trypsin

d. Asp-N-protease

2. Deficiency of which of the following enzymes leads to reduction in sperm production?

a. Vitamin A

b. Vitamin D

c. Vitamin E

d. Vitamin K

3. The conversion of Fructose-6-phopshate to Fructose-1,6-bisphosphate is catalyzed by which one of the following enzymes?

a. Phosphofructokinase 1

b. Phosphofrucokinase 2

c. Fructose-1,6-Bisphosphatase-1

d. Fructose-1,6-Bisphosphatase-2

4. Coenzyme Q is directly involved in electron transport chain as:

a. a water soluble electron donor

b. a lipid soluble electron donor

c. a covalently attached cytochrome cofactor d. directly to oxygen

5. The terminal electron acceptor during mitochondrial respiration:

a. H2O

b. NAD+

c. ATP d. O2

6. Cytochrome oxidase contains which of the following metal ions, as co-factor?

a.Co

b. Mg

c. Fe d. Hg

7. Crystalline substances dissolve more easily in water because of?

a. increase in entropy as the crystal lattice is distorted

b. increased electrostatic interaction between water and charged molecules

c. decreased electrostatic interaction

d. both a and b

8. The macromolecule structure and function is stabilized as result of these kinds of interaction except one:

a. Van-der Walls interaction

b. Covalent interaction forces

c. Hydrogen bonding

d. Electrostatic forces of interactions

9. Which among the following is true about the properties of aqueous solution?

a. A pH changes from 5.0 to 6.0 reflects an increase in the hydroxide ion concentration (OH-) of 20%

b. A Ph change from 8.0 to 6.0 reflects a decrease in the proton concentration (H+) by a factor of 100

c. Charged molecules are generally insoluble in water

d. Hydrogen bonds form readily in aqueous solution

10. The enzyme fumarase catalyzes the reversible hydration of fumaric acid to L-malate, but it will not catalyze the hydration of maleic acid to the cis isomer of fumaric acid. This is an example of

a. Biological activity

b. Racemization

c. Stereoisomerization

d. Stereospecificity

11. Calculate the molarity of an NaOH in a solution prepared by dissolving 2 grams in water to form 250 mL of the solution.

a. 0.2 M

b. 0.4 M

c. M

d. 0.8 M

12. Which amino acid will frequently plot in the disallowed region of Ramachandran plot?

a. proline

b. Glycine

c. Alanine

d. Glutamine

13. The proline derivative 4-hydroxyproline plays an essential role in folding of collagen and maintaining its structure. The deficiency of which of the following vitamins will lead to a distorted protein folding:

a. Vitamin A

b. Vitamin D

c. Vitamin C

d. Vitamin B

14. A protein has got disulphite linkages. The native protein with intact disulphide linkages is cleaved into small peptides. Which technique can be used for the detection of the number of cysteine residues joined by disulphide linkages?

a. MALDI

b. FAB-MS

c. Gas-chromatographyd. X-ray crystallography

15. In a mixture the presence of glycoproteins can be most efficiently determined by which of the following components

a. Ninhydrin

b. Coomassie blue

c. Lectin

d. Silver nitrate

16. Myoglobin and hemoglobin have very:

a. different primary and tertiary structures

b. similar primary and tertiary structures

c. similar primary structures but different tertiary structures

d. similar tertiary structures but different primary structures

17. Isozymes are:

a. enzymes that differ in amino acid sequences

b. the are formed from different alleles of same gene

c. that are products of different genes d. both (a) and (c)

18. The graph shown below is characteristic of:

a. Competitive inhibition

b. Uncompetitive inhibition

c. Mixed inhibition d. Noncompetitive inhibition

19. Which among the following has got highest bond dissociation energy?

a. C=O

b. C=N

c. C=C

d. C-C

20. The alpha-helix is stabilized by: a. Hydrogen bonds b. Vanderc. Covalent bonds d. Ionic bonds

21. A solution was prepared by mixing NaOH & agar with distilled water. Which of the following species will contribute to changes in pH?

a. NaOH b. Distilled water c. Agar d. Both NaOH and agar

22. What is the effect of adding CaOH)2 is added to distilled water at pHa. pH will change to 8 b. pH will change to 6 c. pH will not change d. pH range of 6 to 7 will be maintained

23. A buffer was prepared by mixing HCl & NaOH. Both the species will help in the formation of conjugate acid base pair. Which of the following will act as conjugate acid- base pair to maintain the buffering range?

a. H+ + OH

b. NaCl, H c. Both a & b d. The buffer cannot be made using HCl & NaOH

24. The temperature of the system .decreases in an a. adiabatic compression b. isothermal expansion c. isothermal compression d. adiabatic expansion

25. In the binding of oxygen to myoglobin, the relationship between the concentration of oxygen and the fraction of binding sites occupied can be best described as: a. Hyperbolic

b. Linear with a negative slope c. Linear with positive slope d. Sigmoidal

26. The hydrolysis of ATP has a large negative ∆G'°; nevertheless it is stable in solution due to: a. entropy of stabilization b. ionization of phosphate c. resonance stabilization d. the hydrolysis reaction has a large activation energy

27. Why Uracil is present only in RNA and not in DNA?

a. It is bulky then Thymine, affecting DNA stability

b. Uracil can easily convert to Cytosine, causing frame shift in DNA

c. Uracil forms 3 hydrogen bonds, hence cannot bind to Adenine in DNA

d. Uracil can easily convert to Cytosine, causing base transition.

28. The DNA duplex absorbs 40% less UV rays than individual bases. Which of the following statement can be a reasonable explanation for the above statement?

a. The duplex formation diminishes the capacity of bass to absorb due to base stacking.

b. The duplex has major and minor grooves

c. The individual bases show different chemical property than that of Duplex form

d. The duplex is present inside the nucleus, where as individual bases in nucleotide pool of cytoplasm

29. Topoisomerases can cut and re knot the DNA strands. They break the phosphodiester bond between 2 adjacent bases. Surprisingly they do not use ATP hydrolysis energy to cleave the high energy Phosphate bond. What may be a possible explanation for this peculiar behavior?

a. The total ATP used to cleave the bond, is regenerated at the end of the reaction, hence net ATP is used is Zero.

b. The phospho diester bonds are not high energy bonds

c. They pull the adjacent bases apart, thus breaking the bond

d. Topoisomerases form phospho-tyrisine bond, which conserves the energy , that is used to form the ester bond to rejoin the DNA

30. Ethidium Ions cause DNA to unwind. They are florescent under UV radiation and used extensively in DNA mobility studies. They intercalate the DNA chaging the normal rotation of base pairs from 36° to 10°. If you happen to add one ethidium ion between every 2 bases of DNA, how many bases you will find in per turn of the helix?

a.36

b.72

c. 10

d. 5.5

31. DNA and RNA are the nucleic acids differing in only one OH- group. The DNA forms major genetic material and RNA , the messenger role and catalytic roles. The trans-esterification reaction involves formation of esters in the ribose sugar. What may be the reason, that this reaction is specific for only RNA , but not DNA.

a. The esterase enzymes are present in the nucleus, where as DNA is present in the nucleus.

b. The DNA has 3’–

c. The DNA has no 3’ OH group

d. The DNA has no 2’OH

32. A non supercoiled B-DNA molecule is composed of 4800bps. How many helical turns are present? a.10

b. 380 c. 480 d. Impossible to determine

33. If RNA in TMV contain 28% cytosine then what percentage of the bases in TMV are adenine?? a.30% b.20%

c. 60%

d. Cannot be determined

34. Where would you expect a polypeptide region that is rich in the amino acids valine, leucine and isoleucine to be located in the folded polypeptide?

a. Interior of polypeptide

b. Exterior of polypeptide

c. In the active site of globular proteins

d. In the DNA degrading ezzyme complex

35. Enzymes that break down the DNA catalyze the hydrolysis of the covalent bonds that join nucleotides together. What would happen to DNA molecules treated with these enzymes?

a. the two strandes of the double helix would separate

b. the phosphodiester linkages of the polynucleotide backbone would be broken.

c. The purines would be separated from the deoxyribose sugars

d. All bases would be separated from the deoxyribose sugar

36. Which one of the following is the correct order of electron transfer in ETC

a. Cytochrome-b; Cytochrome-c; Cytochrome c1; cytochrome (a+a3); Coenzyme Q; Cytochrome b

b. CoenzymeQ; Cytochrome b; Cytochrome C1; Cyrochrome c; Cytochrome (a+ a3)

c. Cytochrome (a + a3); Cytochrome b; Cytochrome c; Cytochrome C1; Coenzyme Q

d. None of the above

37. A person is suffering with cancer. The anticancer drug that has been designed has got to ability to inhibit the uncontrolled glycolytic pathway. Iodoacettae has been used in the treatment. IAA is known as an irreversible inhibitor of all cysteine proteases. Which particular step in glycolysis will be affected upon treatment with IAA?

a. The conversion of 3-phosphoglycerate to 2-phosphoglycerate

b. The conversion of Glyceraldehyde -3-phosphate to 3-phosphoglycerate

c. The conversion of 1,3 Bisphosphoglycerate to 3-phosphoglycerate

d. Phosphoenolpyruvate to Pyruvate

38. In the first step of glycolysis, the enzyme hexokinase uses ATP to transfer a phosphate to glucose to form glucose-6-phosphate. The product continues to be oxidized forming pyruvate in glycolysis and is a precursor to acetyl-CoA for the citric acid cycle. Suppose that a cell has only glucose available for energy and that the activity of hexokinase is suddenly stopped in this cell. Which of the following conditions will occur?

a. The cell will continues to produce energy from mitochondrial electron transport chain

b. The cell will continue producing ATP using citric acid cycle

c. The cell will ultimately be unable to produce ATP

d. The cell will be forced to switch to fermentation to produce ATP

39. During a heart attack, blood flowing to the heart muscle is interrupted by blockage of a coronary artery. Which among the following statement is incorrect with respect to change in metabolism in the heart?

a. oxidative phosphorylation would slow down in the mitochondria

b. the production of water by mitochondria will be increased

c. the rate of production of lactic acid would be stimulated d. the use of glucose by the muscle tissue would increase

40. If you isolate mitochondria and place them in buffer with a low pH they begin to manufacture ATP. Which of the following can be a satisfactory explanation for this behavior?

a. Low pH increases the concentration of base causing mitochondria to pump outH+ to the inter membrane space leading to ATP production.

b. The high external acid concentration causes an increase in H+ in the inter membrane space leading to increased ATP production by ATP synthetase.

c. Low pH increases the acid concentration in the mitochondrial matrix, a condition that normally causes ATP production. d. Low pH increases the OH- concentration in the matrix resulting in ATP production by ATP synthetase

41. Collagen has evolved to provide strength. It is found in connective tissue such as tendons, cartilage, the organic matrix of bone and cornea. The collagen helix is a unique secondary structure quite distinct from the alpha-helix. Which of the following statements is incorrect with respect to collagen structure

a. they contain about 35% glycine, 11% alanine and 21% praline and 4-hydroxyproline

b. three separate polypeptide chains called as alpha chains are supertwisted around each other

c. It is rich in hydrophobic residues like Alanine, valine, leucine, methionine

d. It is left handed and has three amino acid residues per turn

42. Which among the following graph will best represent the ramachandran plot for alanine?

43. The tabulated data given here indicates the results of an enzymatic assay carried out under two different experimental conditions. The value of Vmax and Km for both sets of conditions are:

a. Conditon A: Vmax = 0.28 and Km = 0.42; Condition B: Vmax = 0.36 and Km = 0.52

b. Condition A: Vmax = 0.42 and Km = 0.28; Condition B: Vmax = 0.52 and Km = 0.36

c. Condition A: Vmax = 0.42 and Km = 1.1; Condition B: Vmax = 0.52 and Km = 1.1

d. Condition A: Vmax = 0.40 and Km = 2.0; Condition B: Vmax = 0.51 and Km = 2.0

44. Consider an enzymatic conversion as shown below,

The rate of formation of [ES] complex is 1.5 * 10-5 M-1 s-1, the rate of dissociation of [ES] complex to [E] and [S] is 4.6 * 10-6 s-1, and formation of product is the rate limiting step. The Km for this reaction is

a. 1 M

b. 5 mM

c. 2.5 mM

d. 0.2 M

45. The graph shown below indicates reaction rates versus substrate concentration for three different enzymes. Identify which curve corresponds to which of the three different enzymes.

a. A= Michaelis Menton; B = Allosteric dimer; C = Allosteric octamer

b. A= Allosteric dimer; B = Michaelis Menton; C = Allosteic octamer

c. A = Allostertic octamer; B = Allosteric dimer; C = Michaelis Menton

d. A = Allosteric dimer; B = Allosteric octamer; C = Michaelis Menton

46. The conversion of phosphoenol-pyruvate to pyruvate is highly favourable and is an example of substrate level phosphorylation. The free energy of hydrolysis of phosphoeneol pyruvate is -61.9 kJ/mol. Which among the following statements best explains this observation?

a. The product of hydrolysis is keto pyruvate, which quickly tautomerizes to pyruvate. This keto- enol tautomerization stabilizes the products of hydrolysis relative to phosphoenolpyruvate, which cannot tautomerize.

b. The product of hydrolysis is enol pyruvate, which quickly tautomerizes to pyruvate. This keto- enol tautomerization stabilizes the products of hydrolysis relative to phosphoenolpyruvate, which cannot tautomerize.

c. The product of hydrolysis is enol pyruvate, which does not quickly tautomerizes to pyruvate. This keto- enol tautomerization stabilizes the products of hydrolysis relative to phosphoenolpyruvate, which cannot tautomerize.

d. The product of hydrolysis is enol pyruvate, which quickly tautomerizes to pyruvate. This keto- enol tautomerization does not stabilizes the products of hydrolysis relative to phosphoenolpyruvate, which cannot tautomerize.

47. Insulin is a peptide hormone produced in the pancreas by beta cells. It aims to lower the glucose level in blood and is normally secreted after every meal. Their action is to activate transporters of glucose found on the surface of the cells, so that the cells can take up glucose from blood and into cells. What glucose transporter is regulated by the action of insulin?

a. Sodium-Glucose transporter (SGLT)

b. Glucose transporter2 (GLUT 2)

c. Glucose transporter 5 (GLUT 5)

d. Glucose transporter 4 (GLUT 4)

48. Which among the following statements in incorrect about myoglobin the oxygen carrying molecule in muscles?

a. It is a single polypeptide chain made up of 153 amino acids

b. The interior consists of myoglobin consists of nonpolar residues such as leucine, valine, methionine, phenylalanine

c. Histidine residue plays a crutial role in binding iron and oxygen

d. They consist of many charged residues like aspartate, glutamate, lysine, arginine

49. The first step in two-dimensional gel electrophoresis generates a series of protein bands by isoelectric focusing. In a second step, a strip of this gel is turned 90 degrees, placed on gel containing SDS, and electric current is applied. In this second step:

a. proteins with similar isoelectric points become further separated according to their molecular weights b. the visual bands become stained so that the isoelectric focus pattern can be visualized c. the individual bands become visualized by interacting with protein-specific antibody in the second gel d. the protein in the bands separate more completely because the second electric current in the opposite polarity to the first current

50. In the diagram below, the plane drawn behind the peptide bond indicates:

a. plane of raotation around C b. region of steric hindrance determined by the large C=O group c. absence of rotation around the C-N bond because of its partial double-bond character d. theoretical space between -180 and +180 degrees that can be occupied by the peptide bonds

51. Experiments on denaturation and renaturation after the reduction and reoxidation of the in the enzyme ribonuclease (RNase) have shown that:

a. the completely unfolded enzyme, with all b. the enzyme, dissolved in water, is thermodynamically stable relative to the mixture of amino acids whose residues are contained in RNase

c. the primary sequence of RNase is sufficient to determine its specific secondary and tertiary structure d. native ribonuclease does not have a unique secondary and tertiary structure

52. Which of the following statements about the glycerol-phosphate/malate-aspartate shuttles is true?

a. The glycerol-phosphate shuttle transports NADH across the outer mitochondrial membrane, while the malate-aspartate shuttle transports NADH across the inner mitochondrial membrane

b. The malate-aspartate shuttle is a less energy-efficient means of transporting metabolites across the mitochondrial membrane c. FAD serves as the oxidizing agent in the mitochondrial matrix with the glycerol-phospate shuttle. d. None of these are true

53. Dinitrophenol is an inhibitor that decreases the production of ATP by a. incorporating into the inner mitochondrial membrane thereby making the membrane permeable to protons b. binding a proton on the acidic side of the membrane, diffusing through the membrane, and releasing the proton on the alkaline side of the membrane. c. binding to the T state of F1 of ATP synthase thereby inhibiting the conformational change necessary to form ATP d. stimulating proton flux through the uncoupling protein, thermogenin.

54. Both water and glucose share an -OH that can serve as a substrate for a reaction with the terminal phosphate of ATP catalyzed by hexokinase. Glucose, however, is about a million times more reactive as a substrate than water. The best explanation is that:

a. the larger glucose binds better to the enzyme; it induces a conformational change in hexokinase that brings active-site amino acids into position for catalysis.

b. water normally will not reach the active site because it is hydrophobic

c. water and the second substrate, ATP, compete for the active site, resulting in a competitive inhibition of the enzyme

d. the -OH group of water is attached to an inhibitory H atom while the glucose -OH group is attached to C

55. Lysozyme has a pH centered around pH 5.0. The active site of lysozyme contains a glutamic acid residue (pKa = 5.5) and an aspartic acid residue (pKa = 4.0). Which of the following statements is correct about the mechanism of lysozyme?

a. The glutamic acid residue is in a more polar environment than the aspartic acid.

b. During the entire catalytic mechanism, the aspartic acid residue remains unprotonated.

c. During the mechanism, an oxyanion transition state forms.

d. The glutamic acid residue acts as a general base catalyst.

56. A nonapeptide was determined to have the following amino acid composition: (Lys)2,(Gly)2,(Phe)2,His,Leu,Met. The native peptide was incubated with 1-fluoro-2,4-dinitrobenzene (FDNB) and then hydrolyzed; 2,4-dinitrophenylhistidine was identified by HPLC. When the native peptide was exposed to cyanogen bromide (CNBr), an octapeptide and free glycine were recovered. Incubation of the native peptide with trypsin gave a pentapeptide, a tripeptide, and free Lys. 2,4-Dinitrophenylhistidine was recovered from the pentapeptide and 2,4-Dinitrophenylphenylalanine was recovered from the tripeptide. Digestion with the enzyme pepsin produced a dipeptide, a tripeptide and a tetrapeptide. The tertrapeptide was composed of (Lys)2, Phe and Gly. The native sequence was determined to be:

a. Gly-Phe-Lys-Lys-Gly-Leu-Met-Pheb. His Phe

c. His-Leu-Phe-Gly-Lys-Lys-Phe-Met-Gly d. His-Leu-

57. Succinate dehydrogenase catalyzes the conversion of succinate to fumarate. The reaction is inhibited by malonic acid, which resembles succinate but cannot be acted upon by succinate dehydrogenase. Increasing the ratio of succinate to malonic acid reduces the inhibitory effect of malonic acid. Based on this information, which of the following is correct?

a. Succinate dehydrogenase is the enzyme, and fumarate is the substrate.

b. Succinate dehydrogenase is the enzyme, and malonic acid is the substrate.

c. Succinate is the substrate, and fumarate is the product.

d. Fumarate is the product, and malonic acid is a noncompetitive inhibitor.

58. Protein kinases are enzymes that catalyze phosphorylation of target proteins at specific sites, whereas protein phosphatases catalyze removal of phosphate(s) from phosphorylated proteins. Phosphorylation and dephosphorylation can function as an on-off switch for a protein's activity, most likely through

a. the change in a protein's charge leading to a conformational change.

b. the change in a protein's charge leading to cleavage.

c. a change in the optimal pH at which a reaction will occur.

d. a change in the optimal temperature at which a reaction will occur.

59. Which amino acid residue is most likely to be found in the interior of a water soluble globular protein?

a. Ser b. Val

c. Arg

d. Asp

60. Which of the following statements about protein secondary structure is correct?

a. An alpha-helix is primarily stabilized by ionic interactions between the side chains of the amino acids

b. Beta-sheets exist only in antiparellel form

c. Beta-turns often contain proline

d. An alpha-helix can be composed of more than one polypeptide chain.

61. Given below are four enzymatic reactions involved in glycolysis. In which of the following steps is ATP generated?

a. 2-phosphoglycerate to phosphoenol pyruvate

b. Glucose-6-phosphate to fructose-6-phosphate

c. Phosphoenol pyruvate to pyruvate

d. Glyceraldehyde-3-phosphate to 1,3-bisphosglycerate.

62. Which of the following compounds is a positive allosteric regulator of the enzyme pyruvate carboxylase?

a. ATP

b. acetyl CoA

c. Biotin

d. PEP

63. Which one of the following sequences in proteins corresponds to N-glycosylation site?

a. Asn-Ser/Thr

b. Asn-X-Ser/Thr

c. Asn-X-X-Ser/Thr

d. Ser/Thr-Asn

64. Competitive inhibitors inhibit biochemical reactions in such a way as to seemingly:

a. Increase the concentration of substrate. b. Reduce the concentration of enzyme. c. Increase the concentration of enzyme. d. Reduce the concentration of substrate.

e. Denature the enzyme

65. Penicillin is a drug that acts by:

a. Irreversibly inhibiting transpeptidase. b. Reversibly inhibiting transpeptidase. c. Competitively inhibiting transpeptidase. d. Noncompetitively inhibiting transpeptidase. e. None of these.

66. In proteins under normal physiological condition (pH =7) which of the following amino acids will be partially ionized?

a. Arginie

b. Lysine

c. Histidine

d. Serine

67. Active site of Topoisomerase I contains

a. Tyrosine

b. Tryptophan

c. Glycine

d. Fe+3 ions

68. R-groups in an

a. Extend outward from the central axis to avoid interfering stearically with each other b. Extend inward from the central axis c. Have to be close together to stabilize the helix d. Form the anti-parallel β-sheets

e. Are hydrogen bonded to peptide bonds four amino acids away in the chain

69. Nonregular, nonrepetitive secondary structures

a. Include α-helices and β-sheets

b. Are bends, loops, and turns that do not have a repeating element

c. Are beta-strands

d. Are Beta-sheets

e. Include beta-sheets

70. What is the axial ratio (length: diameter) of a viral DNA molecule 20μm long?

a. 1 x 105

b. 1 x 104

c. 2 x 104

d. 2 x 105

71. Why RNA is hydrolyzed by alkali, whereas DNA is not?

a. RNA has Uracil, unlike DNA

b. The 2’deoxy sugar of RNA is more susceptible than 2’ oxy ribose of DNA

c. The 2’deoxy sugar of DNA is less susceptible than 2’ oxy ribose of RNA

d. The 2’ Deoxy ribose of DNA is not affected by alkali as DNA is present inside the Nucleus and wrapped by nucleosomes, so that no DNA is free for alkali action.

72. Which of the following statement is true?

A. Chromosomes are membrane bound and are stable in entire life span of a cell.

B. If barr body is formed in female, no genes form father will be expressed in the child.

a. Only A

b. Only B

c. Both A and B

d. None of these.

73. Calculate the specificity constant for an enzyme if its kcat = 1.4 x 104 s-1 Km = 90 µM. Is this enzyme very efficient?

a. 16X 10

b. 16X10

c. 16X10

d. 1.6X 10

74. Which of the following statements about the mechanism of the catalytic triad of chymotrypsin is correct?

a. A proton moves from the serine to the histidine to the aspartate side chain in the catalytic triad of chymotrypsin.

b. A proton moves from the aspartate to the serine to the histidine side chain in the catalytic triad of chymotrypsin.

c. A proton moves from the serine to the histidine side chain in the catalytic triad of chymotrypsin.

d. A proton moves from the aspartate to the histidine side chain in the catalytic triad of chymotrypsin.

75. Electromotive force (e.m.f) is a measure of charge of solution. When we dip a electrodes in solutions, they pick up some electric charges from the solution. The charge development depends on its pH content. In a saturated calomel electrode pH = [e.m.f - 0.246]/ 0.059. When the electrodes are dipped in a solution the voltmeter reads 0.652. What is the pH if the solution?

a. 6.9

b. 6.0

c. 9.6

d. 0.69

76. A protein contains 2 Trp and 4 Tyr residues. The molecular mass of the protein is 17000D and that of Trp and Tyr are 204 and 180 D respectively. Values of E1%cm, the absorption coefficient of 1% (g/v) solutions of Trp and Tyr in 1-cm cell at 280 nm, are 269.60 and 83.33, respectively. The absorption of 1mg/ml protein solution in 1cm-cell at 280nm will be:

a. 0.1

b. 1.0

c. 0.7

d. 1.7

77. -5, the entropy of the reaction has not changed, and the reaction is just a isomerization step. What is the enthalpy/ bond energy of the isomerization step? ( R = 8.314 J/mol.K ; T = 25C )

a. 24 KJ/mol

b. 50Kj/mol

c. 24 J /mol

d. 0.24 kj/mol.

78. If a 0.1 M solution of glucose 1-phosphate is incubated with a catalytic amount of phospho- glucomutase, the glucose 1-phosphate is transformed to glucose 6-phosphate until equilibrium is reached. At equilibrium, the concentration of glucose 1-phosphate is 4.5 x 10–3 M and that of glucose 6phosphate is 8.6 x 10 2 M. What is the value of ∆G'° for this reaction?(in the direction of glucose 6-phosphate formation). (R = 8.315 J/mol•K; T = 298 K)

a. 8.3 kJ/mol

b. +7.3 kJ/mol

c. 7.3 kJ/mol

d. +8.3 kJ/mol

79. Which of the following graphs shows the results of reaction rate vs substrate concentration for an non-allosteric enzyme in the absence and presence of a non-competitive inhibitor (non-competitive inhibitors bind to an enzyme at a site different than the active site)?

a. 1

b. 2

c. 3

d. 4

e. none of the graphs.

80. When a mixture of glucose 6-phosphate and fructose 6-phosphate is incubated with the enzyme phosphohexose isomerase, the final mixture contains twice as much glucose 6-phosphate as fructose 6-phosphate. Which one of the following statements is most nearly correct, when applied to the reaction below (R = 8.315 J/mol•K and T = 298 K)?

Glucose 6-phosphate

a. ∆G'° is +1.7 kJ/mol.

b. ∆G'° is

c. ∆G'° is incalculably large and negative.

d. ∆G'° is incalculably large and positive.

e. ∆G'° is zero.

81. Given that the standard free energy change (∆G°) for the hydrolysis of ATP is –7.3 K cal/mol and that for the hydrolysis of Glucose 6-phosphate is 3.3 Kcal/mol, the ∆G° for the phosphorylation of glucose is Glucose + ATP

a. 10.6 Kcal/mol

b. 7.3 Kcal/mol

c. 4.0 Kcal/mol

d. +4.0 Kcal/mol.

82. For the following reaction, ∆G'° = +29.7 kJ/mol.

L-Malate + NAD+  oxaloacetate + NADH + H+. The reaction as written:

a. can never occur in a cell.

b. can occur in a cell only if it is coupled to another reaction for which ∆G'° is positive.

c. can occur only in a cell in which NADH is converted to NAD+ by electron transport.

d. cannot occur because of its large activation energy.

e. may occur in cells at some concentrations of substrate and product.

83. A drug which prevents uric acid synthesis by inhibiting the enzyme xanthine oxidase is

a. Aspirin

b. Allopurinol

c. Colchicine

d. Probenecid

84. Lactate formed in muscles can be utilized through a. Rapoport-Luebeling cycle

b. Glucose-alanine cycle

c. Cori’s cycle

d. Citric acid cycle

85. Which one of the following statements concerning glucose metabolism is correct?

a. The conversion of Glucose to lactate occurs only in the R.B.C

b. Glucose enters most cells by a mechanism in which Na+ and glucose are co-transported

c. Pyruvate kinase catalyses an irreversible reaction

d. An elevated level of insulin leads to a decreased level of fructose 2, 6-bisphosphate in hepatocyte

86. A ketogenic aminoacid is

a. Valine

b. Cysteine

c. Leucine

d. Threonine

87. In abetalipoproteinemia, the biochemical defect is in a. Apo-B synthesis

b. Lipoprotein lipase activity

c. Cholesterol ester hydrolase d. LCAT activity.

88. Conversion of inosine monophosphate to xanthine monophosphate is catalysed by a. IMP dehydrogenase

b. Formyl transferase

c. Xanthine-guanine phosphoribosyl transferase d. Adenine phosphoribosyl transferase

89. Methionine is synthesized in human body from a. Cysteine and homoserine b. Homocysteine and serine c. Cysteine and serine

d. None of these

90. Hydroxylation of phenylalanine requires all of the following except a. Phenylalanine hydroxylase

b. Tetrahydrobiopterin

c. NADH

d. Molecular oxygen

91. The amino acid that undergoes oxidative deamination at significant rate is

a. Alanine

b. Aspartate

c. Glutamate

d. Glutamine

92. All the following statements about phenylketonuria are correct except a. Phenylalanine cannot be converted into tyrosine b. Urinary excretion of phenylpyruvate and phenyllactate is increased c. It can be controlled by giving a lowphenylalanine diet

d. It leads to decreased synthesis of thyroid hormones, catecholamines and melanin

93. Acetyl CoA carboxylase regulates fatty acid synthesis by which of the following mechanism?

a. Allosteric regulation

b. Covalent modification

c. Induction and repression

d. All of these

94. β-Oxidation of fatty acids requires all the following coenzymes except a.CoA

b. FAD

c. NAD d. NADP

95. Which of the following can be oxidized by

a. Saturated fatty acids

b. Monosaturated fatty acids

c. Polyunsaturated fatty acids

d. All of these

96. Propionyl CoA is formed on oxidation of

a. Monounsaturated fatty acids

b. Polyunsaturated fatty acids

c. Fatty acids with odd number of carbon atoms

d. None of these

97. The glycosidic bond between sugar and base is broken by phosphate in both pyrimidine and purine catabolism. Why is this reaction not a problem during pyrimidine biosynthesis; that is, why doesn't the reverse of the reaction catalyzed by orotate phosphoribosyl transferase readily occur?

a. Pyrophosphate is not reactive enough to break glycosidic bonds; only phosphate is.

b. The phosphorolysis reaction can only occur with a nucleoside, not a nucleoside monophosphate.

c. Orotate phosphoribosyl transferase produces pyrophosphate, and pyrophosphate is quickly degraded to phosphate.

d. The carboxylate on orotate inhibits phosphorolysis.

98. Which of the following enzymes or enzyme combinations might allow an organism to avoid incorporating deoxyuridine into its genome?

a. A nucleoside kinase that could phosphorylate any nucleoside to a nucleoside monophosphate.

b. A nucleoside monophosphate kinase and a nucleoside diphosphate kinase that were specific for deoxyuridine nucleosides and nucleotides and did not use deoxythymidine nucleosides and nucleotides.

c. A ribonucleotide reductase that was not allosterically regulated, allowing all nucleoside diphosphates to be reduced equally well.

d. A nucleoside diphosphate kinase that did not use deoxyuridine nucleotides, and deoxyuridine diphosphate phosphatase

99. Mutants in adenosine deaminase cause T lymphocytes and B lymphocytes to develop improperly and lead to severe immune deficiencies. A plausible explanation for this is:

a. Since adenosine is not broken down, RNA and DNA contain too much adenosine.

b. Since adenosine is not broken down, too much ATP is produced and the cell's energy balance is thrown off.

c. Since deoxyadenosine is not broken down, ribonucleotide reductase is down-regulated and too few deoxynucleotides are produced.

d. Since deoxyadenosine is not broken down, purine biosynthesis and catabolism are stimulated, leading to the deposition of uric acid (similar to gout).

100.Glutamate dehydrogenase operates at an important intersection of carbon and nitrogen metabolism. An allosteric enzyme with six identical subunits, its activity is influenced by a complicated array of allosteric modulators. Mutation that alter the allosteric binding site that cause permanent activation of enzyme that lead to a human genetic disorder called hyperinsulinism-hyperammonemia is

a. A mutation in negative modulator GTP.

b. A mutation in positive regulator ADP

c. A mutation in negative regulator GDP

d. A mutation in positive regulator ATP

Glossary Glossary

Acetyl CoA: A metabolic intermediate produced through catabolism of many compounds, including fatty acids, and used as the initial substrate for the central respiratory pathway, the TCA cycle.

Acid: A molecule that is capable of releasing a hydrogen ion.

Acid hydrolases: Hydrolytic enzymes with optimal activity at an acid pH.

Activation energy: The minimal kinetic energy needed for a reactant to undergo a chemical reaction.

Active site: The part of an enzyme molecule that is directly involved in binding the substrate.

Adenosine triphosphate (ATP): Nucleotide consisting of adenosine bonded to three phosphate groups; it is the principal immediateenergy source for prokaryotic and eukaryotic cells.

Aerobes: Organisms dependent on the presence of oxygen to metabolize energy-rich compounds.

Alleles: Alternate forms of the same gene.

Allosteric modulation: Modification of the activity of an enzyme through interaction with a compound that binds to a site (i.e., allosteric site) other than the active site.

Alpha () helix: One possible secondary structure of polypeptides, in which the backbone of the chain forms a spiral (i.e., helical) conformation.

Amide bond: The chemical bond that forms between carboxylic acids and amines (or acidic and amino functional groups) while producing a molecule of water.

Amino acids: The monomeric units of proteins; each is composed of three functional groups attached to a central, group, a defining side chain, and a carboxyl group.

Amphipathic: The biologically important property of a molecule having both hydrophobic and hydrophilic regions.

Amphoteric: Structural property allowing the same molecule to act as an acid or as a base.

Anabolic pathway: A metabolic pathway resulting in the synthesis of relatively complex products.

Anaerobes: Organisms that utilize energy-rich compounds through oxygen-independent metabolic pathways such as glycolysis and fermentation.

Angstrom: The unit, equivalent to 0.1 nm, used to describe atomic and molecular dimensions.

Anion: An ionized atom or molecule with a net negative charge.

Artifact: A structure seen in a microscopic image that results from the coagulation or precipitation of materials that had no existence in the living cell.

Assay: Some identifiable feature of a specific protein, such as the catalytic activity of an enzyme, used to determine the relative

Atomic force microscope (AFM): A high-resolution scanning instrument that is becoming increasingly important in nanotechnology and molecular biology. The AFM operates by scanning a delicate probe over the surface of the specimen.

ATP synthase The ATP-synthesizing enzyme of the inner mitochondrial membrane, which is composed of two chief basal piece, the latter of

Autoradiography: A technique for visualizing biochemical processes by allowing an investigator to determine the location of radioactively labeled materials within a cell. Tissue sections containing radioactive isotopes are covered with a thin layer of photographic emulsion, which is exposed by radiation emanating from the tissue. Sites in the cells containing radioactivity are revealed under the microscope by silver grains after development of the overlying emulsion.

Autotroph: Organism capable of surviving on CO2 as its principal

Base: Any molecule that is capable of accepting a hydrogen ion.

Base composition analysis: The relative amounts of each base in

)

pleated sheet: One possible secondary structure of a polypeptide, in which several -strands lie parallel to each other, creating the conformation of a sheet.

Beta () strand: One possible secondary structure of a polypeptide, in which the backbone of the chain assumes a folded (or pleated) conformation.

Biochemicals: Compounds synthesized by living organisms.

Bioenergetics: The study of the various types of energy transformations that occur in living organisms.

Biosynthetic pathway (secretory pathway): Route through the cytoplasm by which materials are synthesized in the endoplasmic

reticulum or Golgi complex, modified during passage through the Golgi complex, and transported within the cytoplasm to various destinations such as the plasma membrane, a lysosome, or a large vacuole of a plant cell. The alternative term secretory pathway has been used because many of the materials synthesized in the pathway are destined to be discharged (secreted) outside the cell.

Buffers: Compounds that can interact with either free hydrogen or hydroxyl ions, minimizing a change in pH.

Carbohydrates: (Glycans) Organic molecules including simple sugars (monosaccharides) and multisaccharide polymers, which largely serve as energy-storage and structural compounds in cells.

Catabolic pathway: A metabolic pathway in which relatively complex molecules are broken down into simpler products.

Cation: An ionized atom or molecule with an extra positive charge.

Cellulose: Unbranched glucose polymer with assembles into cables and serves as a principal structural element of plant cell walls.

Chaperones: Proteins that bind to other polypeptides, preventing their aggregation and promoting their folding and/or assembly into multimeric proteins.

Chaperonins: Members of the Hsp60 class of chaperones, e.g., GroEL, that form a cylindrical complex of 14 subunits within which the polypeptide folding reaction takes place.

Chemiosmotic mechanism: The mechanism for ATP synthesis whereby the movement of electrons through the electron-transport chain results in establishment of a proton gradient across the bacterial, thylakoid, or inner mitochondrial membrane, with the gradient acting as a high-energy intermediate, linking oxidation of substrates to the phosphorylation of ADP.

Chemoautotroph: An autotroph that utilizes the energy stored in inorganic molecules (such as ammonia, hydrogen sulfide, or nitrites) to convert CO2 into organic compounds.

Cholesterol: Sterol found in animal cells that can constitute up to half of the lipid in a plasma membrane, with the relative proportion in any membrane affecting its fluid behavior.

Coactivators: The intermediaries that help bound transcription factors stimulate the initiation of transcription at the core promoter.

Coenzyme: An organic, nonprotein component of an enzyme.

Cofactor The nonprotein component of an enzyme, it can be either inorganic or organic.

Competitive inhibitor: An enzyme inhibitor that competes with substrate molecules for access to the active site.

Conjugate acid: Paired form created when a base accepts a proton in an acid-base reaction.

Conjugate base: Paired form created when an acid loses a proton in an acid-base reaction.

Conjugated protein: Protein linked covalently or noncovalently to substances other than amino acids, such as metals, nucleic acids, lipids, and carbohydrates.

Conserved sequences: Refers to the amino acid sequences of particular polypeptides or the nucleotide sequences of particular nucleic acids. If two sequences are similar to one another, i.e., homologous, they are said to be conserved, which indicates that they have not diverged very much from a common ancestral sequence

Constitutive: Occurs in a continual, non-regulated manner. Can relate to a normal process, such as constitutive secretion, or the result of a mutation that leads to a breakdown in regulation, which causes continual activity, such as the constitutive activation of a signaling

Copper atoms of the electron transport chain: A type of electron carrier; these atoms are located within a single protein complex of the inner mitochondrial membrane that accept and donate a single states.

Covalent bond: The type of chemical bond in which electron pairs are

Cytochromes: A type of electron carrier consisting of a protein bound

Dalton: A measure of molecular mass, with one dalton equivalent to H atom).

Dehydrogenase: An enzyme that catalyzes a redox reaction by

Denaturation: Separation of the DNA double helix into its two

Denaturation: The unfolding or disorganization of a protein from its

Deoxyribonucleic acid (DNA): A double-stranded nucleic acid composed of two polymeric chains of deoxyribose-containing nucleotides. The genetic material of all cellular organisms. DNA may be duplicated as in DNA replication and produced in large quantities of a specific segment as in DNA cloning.

Disulfide bridge: Forms between two cysteines that are distant from one another in the polypeptide backbone or in two separate polypeptides. They help stabilize the intricate shapes of proteins.

DNA gyrase: A type II topoisomerase that is able to change the state of supercoiling in a DNA molecule by relieving the tension that builds up during replication. It does this by traveling along the DNA and

acting like a “swivel,” changing the positively supercoiled DNA into negatively supercoiled DNA.

Dolichol phosphate: Hydrophobic molecule built from more than 20 isoprene units that assembles the basal, or core, segment of carbohydrate chains within glycoproteins.

Domain: A region within a protein (or RNA) that folds and functions in a semi-independent manner.

Electrochemical gradient: The overall difference in electrical charge and in solute concentration that determines the ability of an electrolyte to diffuse between two compartments.

Electrogenic: Any process that contributes directly to a separation of charge across a membrane.

Electron-transfer potential: The relative affinity for electrons, such that a compound with a low affinity has a high potential to transfer one or more electrons in a redox reaction (and thus act as a reducing agent).

Electron-transport or respiratory chain: Membrane-embedded electron carriers that accept high-energy electrons and sequentially lower the energy state of the electrons as they pass through the chain, with the net results of capturing energy for use in synthesizing ATP or other energy-storage molecules.

Electronegative atom: The atom with the greater attractive force; the atom that can capture the major share of electrons of a covalent bond.

Endergonic reactions: Reactions that are thermodynamically unfavorable and cannot occur spontaneously, possessing a

Endoplasmic reticulum (ER): A system of tubules, cisternae, and vesicles that divides the fluid content of the cytoplasm into a luminal space within the ER membrane and a cytosolic space outside the membranes.

Endothermic reactions: Those gaining heat under conditions of constant pressure and volume.

Energy: The capacity to do work, it exists in two forms: potential and kinetic.

Enthalpy change (ΔH): The change during a process in the total energy content of the system.

Entropy (S): A measure of the relative disorder of the system or universe associated with random movements of matter; because all movements cease at absolute zero (0 K), entropy is zero only at that temperature.

Enzyme inhibitor: Any molecule that can bind to an enzyme and decrease its activity, classified as noncompetitive or competitive based on the nature of the interaction with the enzyme.

Enzyme substrate complex: The physical association between an enzyme and its substrate(s), during which catalysis of the reaction takes place.

Enzymes: The vitally important protein catalysts of cellular reactions.

Equilibrium constant of a reaction (Keq): The ratio of product concentrations to reactant concentrations when a reaction is at equilibrium.

Ester bond: The chemical bond that forms between carboxylic acids and alcohols (or acidic and alcoholic functional groups) while producing a molecule of water.

d state: Electron configuration of a molecule after absorption of a photon has energized an electron to shift from an inner to an outer

onic reactions: Reactions those are thermodynamically

uclease: A DNA- or RNA-digesting enzyme that attaches to either the 5’ or 3’ end of the nucleic acid strand and removes one nucleotide

ermic reactions: Those releasing heat under conditions of

: Molecules consisting of a glycerol backbone linked by ester bonds to three fatty acids, also termed triacylglycerols.

acid: Long, unbranched hydrocarbon chain with a single

back inhibition: A mechanism to control metabolic pathways where the end product interacts with an enzyme in the pathway,

tation: An anaerobic metabolic pathway in which pyruvate is converted to another molecule (often lactate or ethanol, depending on the organism) and NAD+ is regenerated for use in glycolysis.

law of thermodynamics: The law of conservation of energy, which states that energy can neither be created nor destroyed.

Flavoproteins: A type of electron carrier in which a polypeptide is bound to one of two related prosthetic groups, either FAD or FMN.

Free energy change: (ΔG) The change during a process in the amount of energy available to do work.

Free radical: Highly reactive atom or molecule that contains a single unpaired electron.

Functional groups: Particular groupings of atoms that tend to act as a unit, often affecting the chemical and physical behavior of the larger organic molecules to which they belong.

Genes: In nonmolecular terms, a unit of inheritance that governs the character of a particular trait. In molecular terms, a segment of DNA containing the information for a single polypeptide or RNA molecule, including transcribed but non-coding regions.

Genetic code: Manner in which the nucleotide sequences of DNA encode the information for making protein products.

Glycogen: Highly branched glucose polymer that serves as readily available chemical energy in most animal cells.

Glycolysis: The first pathway in the catabolism of glucose, it does not require oxygen and results in the formation of pyruvate.

Glycosidic bond: The chemical bond that forms between sugar molecules.

Glycosylation: The reactions by which sugar groups are added to proteins and lipids.

Glycosyltransferases: A large family of enzymes that transfer specific sugars from a specific donor (a nucleotide sugar) to a specific receptor (typically the growing end of an oligosaccharide chain).

Glyoxysomes: Organelles found in plant cells that serve as sites for enzymatic reactions including the conversion of stored fatty acids to carbohydrate.

Guanosine triphosphate (GTP): A nucleotide of great importance in cellular activities. It binds to a variety of proteins (called G proteins) and acts as a switch to turn on their activities.

Half-life: A measure of the instability of a radioisotope, or, equivalently, the amount of time required for one-half of the radioactive material to disintegrate.

Head group: The polar, water-soluble region of a phospholipid that consists of a phosphate group linked to one of several small, hydrophilic molecules.

Heat shock response: Activation of the expression of a diverse array of genes in response to temperature elevation. The products of these genes, including molecular chaperones, help the organism recover from the damaging effects of elevated temperature.

Hemicelluloses: Branched polysaccharides of the plant cell wall whose backbone consists of one sugar, such as glucose, and sidechains of other sugars, such as xylose.

Heterotroph: Organism that depends on an external source of organic compounds.

Highly repeated fraction: Typically short (a few hundred nucleotides at their longest) DNA sequences that are present in at least 105 copies per genome. The highly repeated sequences typically account for about 10 percent of the DNA of vertebrates.

Histones: A collection of small, well-defined, basic proteins of chromatin.

hnRNP (heterogeneous nuclear ribonucleoprotein): The result of the transcription of each hnRNA which becomes associated with a variety of proteins; hnRNP represents the substrate for the processing reactions that follow.

Hydrocarbons: The simplest group of organic molecules, consisting solely of carbon and hydrogen.

Hydrogen bond: The weak, attractive interaction between a hydrogen atom covalently bonded to an electronegative atom (thus, with a partial positive charge) and a second electronegative atom.

Hydrophilic: The tendency of polar molecules to interact with surrounding water molecules, which are also polar; derived from

Hydrophobic interaction: The tendency of nonpolar molecules to aggregate so as to minimize their collective interaction with ter molecules; derived from “water fearing.”

Induced fit: The conformational change in an enzyme after the substrate has been bound that allows the chemical reaction to

Introns: Those parts of a split gene that correspond to the intervening

Ion: An atom or molecule with a net positive or negative charge because it has lost or gained one or more electrons during a chemical

Ionic bond: A noncovalent bond occurring between oppositely

Iron-sulfur proteins: A group of protein electron carriers with an

Irreversible inhibitor: An enzyme inhibitor that binds tightly, often covalently, thus inactivating the enzyme molecule permanently.

Isoelectric point: The pH at which the negative charges of the component amino acids of a protein equal the positive charges of the

Isoforms: Different versions of a protein. Isoforms may be encoded by separate, closely related genes, or formed as splice variants by alternative splicing from a single gene.

Kinetic energy: Energy released from a substance through atomic or molecular movements.

Lipids: Nonpolar organic molecules, including fats, steroids, and phospholipids, whose common property of not dissolving in water contributes to much of their biological activity.

Macromolecules: Large, highly organized molecules crucial to the structure and function of cells; divided into polysaccharides, certain lipids, proteins, and nucleic acids.

Mass spectrometry: Methodology to identify molecules (including proteins). A protein or mixture of proteins is fragmented, converted into gaseous ions, and propelled through a tubular component of a mass spectrometer, causing the ions to separate according to their mass/charge (m/z) ratio. Identification of the protein(s) is made by comparison with a computer database of the sequence of proteins encoded by a particular genome.

Maximal velocity (Vmax): The highest rate achieved for a given enzymatically catalyzed reaction, it occurs when the enzyme is saturated with substrate.

Membrane potential: The electrical potential difference across a membrane.

Messenger RNA (mRNA): The intermediate molecule between a gene and the polypeptide for which it codes. Messenger RNA is assembled as a complementary copy of one of the two DNA strands that encodes the gene.

Metabolic intermediate: A compound produced during one step of a metabolic pathway.

Metabolic pathway: A series of chemical reactions that results in the synthesis of an end product important to cellular function.

Metabolism: The total of the chemical reactions occurring within a cell.

Methylguanosine cap: Modification of the 5’ end of an mRNA precursor molecule, so that the terminal “inverted” guanosine is methylated at the 7’ position on its guanine base, while t nucleotide on the internal side of the triphosphate bridge is methylated at the 2’ position of the ribose. This cap prevents the 5’ end of the mRNA from being digested by nucleases, aids in transport of the mRNA out of the nucleus, and plays a role in the initiation of mRNA translation.

Michaelis constant (Km) In enzyme kinetics, the value equal to the substrate concentration present when reaction rate is one-half of the maximal velocity.

MicroRNAs (miRNAs): Small RNAs (20-23 nucleotides long) that are synthesized from many sites in the genome and involved in inhibiting translation or increasing degradation of complementary mRNAs.

Mitochondrial matrix: The aqueous compartment within the interior of a mitochondrion. Mitochondrial membranes The outer membrane serves as a boundary with the cytoplasm and is relatively permeable, and the inner membrane houses respiratory machinery in its many invaginations and is highly impermeable.

Mitochondrion: The cellular organelle in which aerobic energy transduction takes place, oxidizing metabolic intermediates such as pyruvate to produce ATP.

Moderately repeated fraction: DNA sequences that are repeated from a few to several hundred thousand times within a eukaryotic

genome. The moderately repeated fraction of the DNA can vary from about 20 to about 80 percent of the total DNA. These sequences may be identical to each other or nonidentical but related.

Molecular chaperones: Various families of proteins whose role is to assist the folding and assembly of proteins by preventing undesirable interactions.

Motif: A substructure found among many different proteins, such as the  barrel, which consists of  strands connected by an -helical region.

Multiprotein complex: The interaction of more than one complete meters.

Nascent protein: A protein in the process of being synthesized, i.e.,

Noncompetitive inhibitor: An enzyme inhibitor that does not bind at the same site as the substrate, and so the level of inhibition depends

Noncovalent bond: A relatively weak chemical bond based on attractive forces between oppositely charged regions within a

Nonpolar molecules: Molecules whose covalent bonds have a nearly symmetric distribution of charge because the component atoms have

Nonrepeated fraction: Those DNA sequences in the genome that are present in only one copy per haploid set of chromosomes. These sequences contain the greatest amount of genetic information including the codes for virtually all proteins other than histones.

Nucleic acid: Polymers composed of nucleotides, which in living organisms are based on one of two sugars, ribose or deoxyribose, yielding the terms ribonucleic acid (RNA) and deoxyribonucleic acid

Nucleic acid hybridization: A variety of related techniques that are based on the fact that two single-stranded nucleic acid molecules of complementary base sequence will form a double-stranded hybrid.

Nucleotide: The monomer of nucleic acids, each consists of three parts: a sugar (either ribose or deoxyribose), a phosphate group, and a nitrogenous base, with the phosphate linked to the sugar at the 5’ carbon and the base at the 1’ carbon.

Oils: Fats that are liquid at room temperature.

Oligosaccharides: Small chains composed of sugars covalently attached to lipids and proteins; they distinguish one type of cell from another and help mediate interactions of a cell with its surroundings.

Operon: A functional complex on a bacterial chromosome comprising a cluster of genes including structural genes, a promoter region, an operator region, and a regulatory gene.

Oxidation: The process through which an atom loses one or more electrons to another atom, in which the atom gaining electrons is considered to be reduced.

Oxidation-reduction (redox) potential: The separation of charge, measured in voltage, for any given pair of oxidizing-reducing agents, such as NAD + and NADH, relative to a standard couple (e.g., H+ and H2 ).

Oxidation-reduction (redox) reaction: One in which a change in the electronic state of the reactants occurs.

Oxidative phosphorylation: ATP formation driven by energy derived from high-energy electrons removed during substrate oxidation in pathways such as the TCA cycle, with the energy released for ATP formation by passage of the electrons through the electrontransport chain in the mitochondrion.

Oxidizing agent: The substance in a redox reaction that becomes reduced, causing the other substance to become oxidized

Peptide bond: The chemical bond linking amino acids in a protein, which forms when the carboxyl group of one amino acid reacts with the amino group of a second amino acid.

pH: The standard measure of relative acidity, it mathematically equals -log[H + ].

Phosphoglycerides: The name given to membrane phospholipids that are built on a glycerol backbone.

Photoautotroph: An autotroph that utilizes the radiant energy of the sun to convert CO2 into organic compounds.

PiwiRNAs (piRNAs): Small RNAs (24-32 bases) that are encoded by a small number of large genomic loci and act to suppress the movement of transposable elements in germ cells. piRNAs are derived from single-stranded precursors and do not require Dicer for processing.

Polar molecules: Molecules with an uneven distribution of charge because the component atoms of various bonds have greatly different electronegativities.

Poly(A) tail: A string of adenosine residues at the 3’ end of an mRNA added posttranscriptionally.

Polyacrylamide gel electrophoresis (PAGE): Protein fractionation technique in which the proteins are driven by an applied current through a gel composed of a small organic molecule (acrylamide) that is cross-linked to form a molecular sieve.

Polypeptide chain: A long, continuous unbranched polymer formed by amino acids joined to one another by covalent peptide bonds.

Potential difference: The difference in charge between two compartments, often measured as voltage across the separating membrane.

Potential energy: Stored energy that can be used to perform work.

Pre-RNA: An RNA molecule that has not yet been processed into its final mature form (e.g., a pre-mRNA, pre-rRNA, or pre-tRNA).

Primary structure: The linear sequence of amino acids within a polypeptide chain.

Primary transcript (or pre-RNA): The initial RNA molecule synthesized from DNA, which is equivalent in length to the DNA from which it was transcribed. Primary transcripts typically have a fleeting existence, ies of “cut-and-

sthetic group: A portion of a protein that is not composed of amino acids, such as the heme group within hemoglobin and myoglobin.

teins: Structurally and functionally diverse group of polymers

teoglycan: A protein-polysaccharide complex consisting of a core protein molecule to which chains of glycosaminoglycans are attached. Due to the acidic nature of the glycosaminoglycans, proteoglycans are capable of binding huge numbers of cations, which in turn draw huge numbers of water molecules. As a result, proteoglycans form a porous, hydrated gel that acts like a “packing”

teome: The entire inventory of proteins in a particular organism,

p): An electrochemical gradient that is built up across energy-transducing membranes (inner mitochondrial membrane, thylakoid membrane, bacterial plasma membrane) following the translocation of protons during electron transport. The energy of the gradient, which is comprised of both a pH gradient and a voltage and is measured in volts, is utilized in the formation of ATP.

rine: A class of nitrogenous base found in nucleotides that has a double-ring structure, including adenine and guanine, which are

Pyrimidine: A class of nitrogenous base found in nucleotides that has a single-ring structure, including cytosine and thymine, which are found in DNA, and cytosine and uracil, which are found in RNA.

Quaternary structure: The three-dimensional organization of a protein that consists of more than one polypeptide chain, or subunit.

rDNA: The DNA sequences encoding rRNA that are normally repeated hundreds of times and are typically clustered in one or a few regions of the genome.

Reannealing (renaturation): Reassociation of complementary single strands of a DNA double helix that had been previously denatured.

Reducing agent: The substance in a redox reaction that becomes oxidized, causing the other substance to become reduced.

Reducing power: The potential in a cell to reduce metabolic intermediates into products, usually measured through the size of the NADPH pool.

Reduction: The process through which an atom gains one or more electrons from another atom, in which the atom losing electrons is considered to be oxidized.

Ribonucleic acid (RNA): A single-stranded nucleic acid composed of a polymeric chain of ribose-containing nucleotides.

Ribosomal RNAs (or rRNAs): The RNAs of a ribosome. rRNAs recognize and bind other molecules, provide structural support, and catalyze the chemical reaction in which amino acids are covalently linked to one another.

Riboswitches: mRNAs that, once bound to a metabolite, undergo a change in their folded conformation that allows them to alter the expression of a gene involved in production of that metabolite. Most riboswitches suppress gene expression by blocking either termination of transcription or initiation of translation.

Ribozyme: An RNA molecule that functions as a catalyst in cellular reactions.

RNA interference (RNAi): A naturally occurring phenomenon in which double-stranded RNAs (dsRNAs) lead to the degradation of mRNAs having identical sequences. RNAi is believed to function primarily in blocking the replication of viruses and restricting the movement of mobile elements, both of which involve the formation of dsRNA intermediates. Mammalian cells can be made to engage in RNAi by treatment of the cells with small (21 nt) RNAs. These small RNAs (siRNAs) induce the degradation of mRNAs that contain the same sequence.

RNA silencing: A process in which small, noncoding RNAs, typically derived from longer double-stranded precursors, trigger sequencespecific inhibition of gene expression.

RNA splicing: The process of removing the intervening DNA sequences (introns) from a primary transcript.

Saturated fatty acids: Those lacking double bonds between carbons.

Second law of thermodynamics: Events in the universe proceed from a state of higher energy to a state of lower energy.

Secondary structure: The three-dimensional arrangement of portions of a polypeptide chain.

Side chain or R group: The defining functional group of an amino acid, which can range from a single hydrogen to complex polar or nonpolar units in the 20 amino acids most commonly found in cells.

Small interfering RNAs (siRNAs): Small (21-23 nucleotide), doublestranded fragments formed when double-stranded RNA initiates the response during RNA silencing.

Small nuclear RNAs (snRNAs): RNAs required for mRNA processing that are small (90 to 300 nucleotides long) and that function in the nucleus.

Small-nucleolar RNAs (snoRNAs): RNAs required for the methylation and pseudouridylation of pre-rRNAs during ribosome formation in the nucleolus.

snoRNPs: (small, nucleolar ribonucleoproteins) Particles that are formed when snoRNAs are packaged with particluar proteins; snoRNPs play a role in the maturation and assembly of ribosomal

snRNPs: Distinct ribonucleoprotein particles contained in spliceosomes, so called because they are composed of snRNAs bound

Specific activity: The ratio of the amount of a protein of interest to the total amount of protein present in a sample, which is used as a

Specificity: The property of selective interaction between components

Spontaneous reactions: Reactions that are thermodynamically favorable, capable of occurring without any input of external energy.

): The change in free energy when one mole of each reactant is converted to one mole of each product under defined standard conditions: temperature of 298 K and

Starch: Mixture of two glucose polymers, amylose and amylopectin, that serves as readily available chemical energy in most plant cells.

Steady state: Metabolic condition in which concentrations of reactants and products are essentially constant, although individual reactions

Stereoisomers: Two molecules that structurally are mirror images of each other and may have vastly different biological activity.

Steroid: Lipid molecule based on a characteristic four-ring hydrocarbon skeleton, including cholesterol and hormones such as testosterone and progesterone.

Structural isomers: Molecules having the same chemical formula but different structures.

Substrate: The reactant bound by an enzyme.

Substrate-level phosphorylation: Direct synthesis of ATP through the transfer of a phosphate group from a substrate to ADP.

Subunit: A polypeptide chain that associates with other chains (subunits) to form a complete protein or protein complex.

tDNA: The DNA encoding tRNAs.

Tandem repeats: A cluster in which a DNA sequence repeats itself over and over again without interruption.

Telomere: An unusual stretch of repeated DNA sequences, which forms a “cap” at each end of a chromosome.

Template: A single strand of DNA (or RNA) that contains the information (encoded as a nucleotide sequence) for construction of a complementary strand

Tertiary structure: The three dimensional shape of an entire macromolecule.

Thermodynamics: Study of the changes in energy accompanying events in the physical universe.

Transfer potential: A measure of the ability of a molecule to transfer any group to another molecule, with molecules having a higher affinity for the group being the better acceptors and molecules having a lower affinity better donors.

Transfer RNAs (tRNAs): A family of small RNAs that translate the information encoded in the nucleotide “alphabet” of an mRNA into the amino acid “alphabet” of a polypeptide.

Transition state: The point during a chemical reaction at which bonds are being broken and reformed to yield products.

Triacylglycerols: Polymers consisting of a glycerol backbone linked by ester bonds to three fatty acids, commonly called fats.

Tricarboxylic acid cycle (TCA cycle): The circular metabolic pathway that oxidizes acetyl CoA, conserving its energy; the cycle is also

Turnover number: The maximum number of substrate molecules that can be converted to product by one enzyme molecule per unit of time.

Ubiquinone: A component of the electron transport chain, ubiquinone is a lipid-soluble molecule containing a long hydrophobic chain

Van der Waals force: A weak attractive force due to transient asymmetries of charge within adjacent atoms or molecules.