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Metabolism MCD Year 1 Anil Chopra

Contents Contents..........................................................................................................................1 Metabolism 1 - Introduction to Protein Structure..........................................................2 Metabolism 2 - Energetics and Enzymes.......................................................................6 Metabolism 3 - Metabolic Pathways and ATP Production I.......................................13 Metabolism 4 -Metabolic Pathways and ATP Production II........................................23 Metabolism 5 - Mitochondria and Oxidative Phosphorylation....................................31 Metabolism 7 - Cholesterol..........................................................................................45 Metabolism 8 - Membrane Trafficking........................................................................53 Metabolism 9 – Integration of Metabolism..................................................................56


Metabolism 1 - Introduction to Protein Structure Anil Chopra 1. Outline the reaction by which amino acids are joined together.

Chirality

Anatomy of an Amino Acid H

H amino group

N H side chain

C R

carboxyl group

C

mirror

OH

Cα R

The whole of the amino acid minus the side chains is known as the backbone.

H

H

α-carbon

Substitutions at the R position or side chain, give rise to the 20 different amino acids e.g. R=CH3 in alanine.

The central Cα carbon atom is a chiral centre (from the Greek, meaning "handed") i.e. it has four different substituents bound to it.

O

NH2 COOH

L-enantiomer

H2N Cα HOOC

R

This gives rise to optical isomers (enantiomers) of each amino acids each of which is a mirror image of the other.

D-enantiomer

Glycine (Gly) has no side chain (only an H atom) and is therefore the only non-chiral amino acid.

Side chains can be polar of non-polar – this is vital to the properties of the protein

Eg. proline – non polar

asparagine - polar

• • • • •

They can also be acidic or basic eg. glutamic acid The state of ionisation of an amino acid provides vital biological properties to many proteins and enzymes, and for this reason cells cannot generally tolerate wide changes in pH. Consequently, If the ionisation state of key amino acids within a protein is altered, a loss of biological activity often results. The ability to take up and release protons gives amino acids some buffering capacity to resist some changes in pH. Individual amino acids are joined in condensation reactions (water is lost) to form peptide chains. 2. Sketch a trimeric peptide, illustrating the amino -terminus, carboxyl terminus and side chains.

2


Anatomy of a Peptide peptide bond

amino terminus

H N H

H

O

C

C

R1

H N

C

H

R2

O C OH

carboxyl terminus

side chains

• • •

The polypeptide chain of a protein rarely forms a disordered structure (random coil) as proteins generally have functions to fulfil, and these functions rely upon specificity. In turn, functionality requires a definite 3D structure or conformation of the polypeptide chain. Proteins generally possess a degree of flexibility necessary for function e.g. muscle fibres 3. Give examples of the post translation modifications of amino acids, with reference to glycosylation, hydroxylation and carboxylation.

• • • • • • • •

Even after synthesis, (post translation), the starting set of 20 amino acids can be modified to create novel amino acids, enhancing the capabilities of the protein. Proline can be modified to produce hydroxyproline e.g. collagen fibres, a major constituent of skin, cartilage, teeth & bones. These additional hydroxyl groups help to stabilise the fibres. The addition of sugar residues to the asparagine residues of proteins (N-linked glycosylation) increases their solubility and also protects them from enzymatic degradation. Deficiency of N-linked sugar chain transfer is detected in congenital carbohydrate-deficient glycoprotein (CGD) syndrome which affects multiple tissues and has life threatening complications. Similarly, g-carboxyglutamate is produced by the carboxylation of glutamate. The formation of g-carboxyglutamate residues within several proteins of the blood clotting cascade (e.g. factor IX) is critical for their normal function by increasing their calcium binding capabilities. The anticoagulant warfarin works by inhibiting the carboxylation reaction. 4. Understand the concepts of primary structure, secondary structure, tertiary structure & quaternary structure with respect to proteins.

Folding of Proteins • • • •

Proteins generally fold into a single conformation of lowest energy. This can occur spontaneous or involve other molecules known as chaperones, which bind to the partly folded polypeptide chain and ensure that the folding continues along the most energetically favourable pathway. By breaking the bonds that hold the protein together, we can denature the protein into the original flexible polypeptide. Common denaturants used within the laboratory are urea (breaks hydrogen bonds) and 2-mercaptoethanol (breaks disulphide bonds). 3


• • • •

Primary structure is the linear sequence of amino acids that make up the protein. Secondary structure is defined as local structural motifs within a protein, e.g. αhelices and β-pleated sheets. Tertiary structure is the arrangement of the secondary structure motifs into compact domains. Quaternary structure is the three dimensional structure of a multimeric protein composed of several subunits 5. Distinguish between a α-helix and a ß-pleated sheet and appreciate the bonds that stabilise their formation.

Neutralisation of the polar groups in amino acids is achieved by their hydrogen bonding to each other in one of two regular structures – α helix and β pleated sheet.

The α helix •

Hydrogen Bonds between the C=O of one residue and the N-H of another residue, 4 amino acids along the helix, stabilise the entire structure.

• •

The side chains of individual amino acids project out from within the a –helix. Although theoretically helices can be either right-handed or left handed, the usage of L-amino acids in proteins means that right-handed helices are favoured. In proline, the last atom of the side chain is bonded to the main chain N atom. This prevents the N atom from hydrogen bonding with the C=O groups of another residue within the helix, thereby distorting the helical conformation, putting a ‘kink’ into it.

• •

The β pleated sheet •

As with the alpha helix, hydrogen bonds between the N-H and C=O groups of two or more b-strands hold the b -pleated sheet sheet together.

4


• • •

In the b-pleated sheet, the NH and C=O groups point out at right angles to the line of the backbone. This almost two dimensional sheet is pleated, like the bellows of an accordion. Alternate b -strands can run in the same direction to give a parallel b-pleated sheet or in opposite directions to give an antiparallel b -pleated sheet. The pleating in each case allows for the best alignment of the hydrogen bonded groups. 6. Appreciate the different types of bond that combine to stabilise a particular protein conformation.

• • •

Covalent bonds (in which two atoms share electrons) are the strongest bonds within protein and exist in the primary structure itself. Covalent bonds can also exist as disulphide bridges. These occur when cysteine side chains within a protein are oxidised resulting in a covalent link between the two amino acids. Hydrogen Bonds occur when two atoms bearing partial negative charges share a partially positively charged hydrogen, the atoms are engaged in a hydrogen bond (H-bond). Ionic interactions arise form the electrostatic attraction between charged side chains e.g. Glu, Asp, Lys and Arg. They are relatively strong bonds, particularly when the ion pairs are within the protein interior and excluded from water. Van der Waals Forces are transient, weak electrostatic attractions between two atoms, due to the fluctuating electron cloud surrounding each atom which has a temporary electric dipole. Although relatively weak and transient in nature, because of the sheer number of these interactions within a protein, they can still have a large part to say in the overall conformation of a protein. Hydrophobic Interactions are a major force driving the folding of proteins into their correct conformation. They juxtapose hydrophobic side chains by packing them into the interior of the protein. This creates a hydrophobic core and a hydrophilic surface to the majority of proteins

Summary • • • • •

Proteins are chains of amino acids linked by peptide bonds which have evolved to fulfil specific functions within the cell. Such functions are reliant upon the 3D structure or conformation of the protein which is held together by a variety of forces. The a-helix and b-pleated sheet are the two staple motifs that define the conformation of a protein. The nature of the amino acid side chain dictates its position within the conformation of the protein. Post-translational modifications of proteins add yet more diversity to protein structure.

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Metabolism 2 - Energetics and Enzymes Anil Chopra 1. Define the 1st and 2nd Laws of thermodynamics. The First Law of Thermodynamics •

Energy can neither be created nor destroyed. i.e. it is simply converted from one form to another.

The Second Law of Thermodynamics • •

• •

In any isolated system, e.g. a single cell or the universe, the degree of disorder can only increase. The amount of disorder in a particular system can be quantified as its entropy. Reactions proceed spontaneously towards products with greater entropy (i.e. more disorder). 2. Explain the concept of free energy and how we can use changes in free energy to predict the outcome of a reaction. However, biological systems are very well ordered. This is achieved by investing taking energy from the environment surrounding the cell and investing it in chemical reactions which maintain order.

At The Single Cell Level

At Single Cell Level

Increased disorder

cell

Increased order

HEAT Surrounding environment

In this scenario, both the 1st and 2nd Laws of thermodynamics are obeyed • •

Entropy changes during a chemical reaction are very difficult to measure. This lead Josiah Gibbs to create the function known as Free Energy. 6


• • • • • •

(Gibb’s) Free Energy is defined as the amount of energy within a molecule that could perform useful work at a constant temperature. It is denoted by the letter G and has units of kilojoules/moles (kJ/mole). The free energy function combines both the 1st and 2nd Laws of thermodynamics. Changes in G (DG) measure the amount of disorder that results from a particular reaction. i.e. In the above scenario, DG measures both the change in order within the cell and also upon the change in entropy of the system. Lets consider the reaction:

A+B

C+D

reactants

products

• • • •

The changes in free energy for this reaction (DG) can be defined by: DG = free energy (C+D) - free energy (A+B) A reaction can only occur spontaneously if DG is negative. Conversely, a reaction cannot occur spontaneously if DG for the reaction is positive. 3. Draw the chemical structure of ATP and explain how it acts as a carrier of free energy and is used to couple energetically unfavourable reactions.

Adenosine Triphosphate (ATP):

• • • • • • •

Phosphoanhydride bonds have a large negative DG of hydrolysis, and are thus said to be "high energy" bonds. ATP ADP + Pi DG°'= - 31 kJ/mole !!! (ΔG°' = standard free energy change at pH 7) Pathways within the cell that synthesise molecules are generally energetically unfavourable e.g. peptide synthesis They take place because they are coupled to an energetically favourable one. Providing that the sum of the DG for the overall reaction is still negative, the reaction will proceed. The majority of energetically unfavourable biochemical reactions rely on the hydrolysis of high-energy phosphate bonds such as those found in ATP. 4. Describe how enzymes act as catalysts of reactions with reference to the reaction catalysed by lysozyme. 7


• • • • • • • •

In a biological setting, most energetically favourable reactions will not occur at a rate useful for life, unless catalysed by enzymes. Enzymes function by lowering the barriers that block a particular reaction. Enzymes bind one or more substrate molecules tightly within a part of the protein known as the active site. Enzymes arrange the substrate(s) in such a way that certain bonds are strained. Key residues within the enzyme participate in either the making or breaking of bonds by altering the arrangement of electrons within the substrate(s). This can often take the form of either oxidation reactions, (in which electrons are removed from a molecule) or reduction reactions (in which electrons are added to a molecule). Since the cellular environment is generally aqueous, often, when a molecule gains an electron, it also simultaneously gains a proton. The transition state is the particular conformation of the substrate in which the atoms of the molecule are rearranged both geometrically and electronically so that the reaction can proceed. Enzymes work by bending their substrates in such a way that the bonds to be broken are stressed and the substrate molecule resembles the transition state. This makes them more amenable to reaction with other molecules. Enzymes function by lowering the barriers that block a particular reaction. Put graphically: Transition State of Substrate

Free energy Substrate

Activation Energy

Substrate

Transition State of Substrate Activation Energy Product

Product

reaction Lysozome • Lysozyme is a component of tears and nasal secretions and is one of the first lines of defence against bacteria. • It catalyses the hydrolysis of sugar molecules within bacterial cell walls that are necessary for their structure. With this bond broken, the bacteria lyse and die. • The activity of lysozyme was discovered by Sir Alexander Fleming, who suffering from a cold, allowed some of his nasal secretions to drip into a bacterial culture. This results in lysed bacteria. • Lysozyme hydrolyzes alternating polysaccharide copolymers of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) which represent the "unit" polysaccharide structure of many bacterial cell walls. • Lysozyme cleaves at the b(1-4) glycosidic linkage, connecting the C1 carbon of NAM to the C4 carbon of NAG. How Lysozome Works 8


• • • • • • • •

Glu35 protonates the oxygen in the glycosidic bond breaking the bond holding the two sugar molecules together. A water molecule enters and is de-protonated by Glu35. Asp52 stabilises the positive charge in the transition state. The hydroxide ion attacks the remaining sugar molecule adding an OH group. Both Glu35 and Asp52 are in their original state to continue catalysis. Glu35 protonates the oxygen in the glycosidic bond breaking the bond holding the two sugar molecules together. A water molecule enters and is de-protonated by Glu35. Asp52 stabilises the positive charge in the transition state. The hydroxide ion attacks the remaining sugar molecule adding an OH group. Both Glu35 and Asp52 are in their original state to continue catalysis. 5. Outline the reaction catalysed by glucose-6-phosphatase and explain what clinical symptoms are linked to inherited deficiencies of this enzyme.

Glucose-6-Phosphatase H2O + Pi Glucose-6-phosphatase Glucose-6-phosphate

• •

Glucose

G-6-Pase is predominantly a liver enzyme that catalyses the above reaction, releasing glucose from the large stores of glycogen within the liver, when blood glucose levels are low. On leaving the liver, the glucose is rapidly taken up by the brain and muscle.

Glucose-6-Phosphatase Deficiency •

• •

A deficiency in G-6-Pase characteristically leads to: - low blood sugar levels - slow growth - large liver - short stature The disease is known as Von Gierke’s disease and sufferers have inherited two mutant copies of the G6Pase gene, one from each parent. Thankfully, only around 1 in 100,000 individuals are affected. 6. Outline the differences between lock and key and induced fit models of substrate-enzyme interactions.

9


Lock and Key Model

+ enzyme

• •

substrate

enzyme-substrate complex

In this model, the shape of the substrate (key) matches that of the active site (lock) of the enzyme. This model explains the specificity of most enzymes for a single substrate.

Induced Fit Model •

In this model, the substrate induces a change in the conformation of the enzyme which results in the formation of the active site. Upon release of products, the enzyme reverts back to its original conformation.

+ enzyme

• •

substrate

enzyme-substrate complex

From crystallographic analysis of enzymes with and without substrate bound, we now hold the induced fit model to be valid. Proteins generally possess a degree of flexibility necessary for function. e.g. muscle fibres 7. Describe graphically, the effects of a) substrate concentration, b) temperature and c) pH on enzyme catalysed reactions.

Substrate Concentration Vmax ½ Vmax

rate of reaction

substrate concentration Km

10


• • • • •

The reaction rates of enzymes vary considerably and can be measured experimentally. This is useful if we are testing an enyme inhibitor e.g. captopril Km is known as the Michaelis Constant ands is defined as the concentration of substrate at which a particular enzyme works at half its maximal velocity. Biochemically, the Km value is useful as a means of comparing the strength of Enzyme-Substrate complexes. Generally a low Km indicates tight binding of a substrate to an enzyme. Conversely, a high Km is indicative of weak binding.

Temperature

rate of reaction

temperature

• •

Chemical reactions speed up as temperature is increased, so, in general, catalysis increases at higher temperatures. However, each enzyme has a temperature optimum, beyond which its conformation is said to be denatured and the enzyme is inactive.

pH Level

rate of reaction

pH

• •

Enzymes have an optimum pH This is due to the catalytic side chains in the active site being in the correct state of ionisation. 8. Illustrate the role of the coenzyme NAD in the reactions catalysed by glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase, referring to the biochemical changes involved in its reduction to NADH 11


NAD+ (Nicotinamide adenine dinucleotide) is a vital component of many dehydrogenation reactions within the body. • It can be described as a coenzyme as it has no catalytic activity of its own and functions only after binding to a enzyme. NAD+ catalyses the dehydrogenation of substrates by readily accepting a hydrogen atom and two electrons. + H+ + 2e-

H- (hydride ion)

NAD+ •

NADH

substrate glyceraldehyde-3-phosphate reaction is oxidised (hydrogen removed) and also phosphorylated in a coupled reaction.

Lactate Dehydrogenase •

During intense exercise, skeletal muscles have to function anaerobically, as oxygen is a limiting factor. As such, the metabolite pyruvate is converted into lactate. This also generates free NAD+ which is needed by the muscle for other reactions. Lactate diffuses from the muscle into the blood stream and is picked up by the liver, where the high levels of NAD+ can be used by lactate dehydrogenase to regenerate pyruvate.

Malate Dehdrogenase •

Malate dehydrogenase oxidises malate to give oxaloacetate, a key metabolite of the TCA cycle.

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Metabolism 3 - Metabolic Pathways and ATP Production I Anil Chopra 1. Sketch a cartoon of the three stages of cellular metabolism that convert food to waste products in higher organisms, illustrating the cellular location of each stage. Metabolism • • •

Digestion – large to small molecules Cellular metabolism I – oxidation of small molecules in cytosol to produce ATP and NADH Cellular metabolism II – oxidation of small molecules in mitochondria

The Three stages of Cellular Metabolism cytosol

Proteins Amino Acids

Glucose

NH2

TCA cycle

ATP

Pyruvate

NADH

Acetyl CoA

Oxidative Phosphoryation

NADH

ATP

H20

O2

Glycolysis

mitochondrion

Simple sugars

CO2 Polysaccharides Fatty Acids &Glycerol

Fats

13


Free Ener gy

Glucose Free energy released as heat

Relatively large activation energy overcome by heat source

CO2 + H20 Reaction

Free energy liberated is invested in carrier molecules such as ATP

Glucose Free Energy

Relatively small activation energies overcome by enzymes and body temperature

CO2 + H20

Reaction Reaction This is around 50% efficient, c.f. car engines which on average are only 20% efficient

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2. Outline the metabolism of glucose by the process of glycolysis, listing the key reactions, in particular those reactions that consume ATP and those that generate ATP. Overview of Glycolysis • 1x 6C glucose 2x 3C pyruvate – 2x ATP produced • Essentially an anaerobic process • Occurs in cytoplasm of cells • Ten reactions that make up glycolysis pathway can be split into two main concepts: -

formation of a high energy compound – involves the investment of energy in the form of ATP susbsequent splitting of compound – produces useful energy in the form of ATP generation

Glycolytic Pathway hexokinase

1.

glucose

glucose-6-phosphate ATP

-

ADP

all kinases transfer a phosphate group reaction irreversible – commits cell to subsequent reactions phosphoglucose isomerase

2.

glucose-6-phostphate -

fructose-6-phosphate

the isomerisation shuffles the glucose chair to give fructose. the logic behind this reaction is that fructose can be split into equal halves when subsequently cleaved phosphofructokinase

3. fructose-6-phosphate bisphosphate

fructose-1,6ATP

-

ADP

here a highly symmetrical, high energy compound is generated regulation of phosphofructokinase exquisitely controls the entry of sugars into the glycolysis pathway adolase

4.

fructose-1,6-bisphosphate

glyceraldehyde-3-phosphate dihydroxyacetone phosphate 15


-

opening of the fructose ring to generate two high energy compounds, one of which, (dihydroxyacetone phosphate) subsequently undergoes isomerisation. triose phosphate isomerase

5. dihydroxyacetone phosphate phosphate -

• •

glyceraldehyde-3-

deficiency in TPI is extremely rare (only 29 documented cases worldwide) since its diagnosis 35 years ago. Most sufferers die within the first 6 years of their lives.

At this half-way point in the pathway one mole of glucose has given rise to two moles of glyceraldehyde-3-phosphate. So far, no energy has yet been produced but two moles of ATP have been used. glyceraldehyde 3-phosphate dehydrogenase

6. 2x glyceraldehyde-3-phosphate bisphosphoglycerate

2x 1,3-

NAD+ + Pi -

NADH

NADH is generated here which can be later used to generate yet more ATP within the mitochondria in a process known as oxidative phosphorylation. phosphoglycerate kinase

7.

2x 1,3-bisphosphoglycerate

2x 3-phosphoglycerate ADP

-

ATP

A phosphate group is transferred to an ADP molecule to give ATP. phosphoglycerate mutase

8.

2x 3-phosphoglycerate -

2x 2-phosphoglycerate

Shuffling of the phosphate group from the 3 to the 2 position enolase

9.

2x 2-phosphoglycerate

2x phosphoenolpyruvate dehydration

pyruvate kinase

10.

2x phosphoenolpyruvate

2x pyruvate 16


ADP •

ATP

transfer of the high energy phosphate group to ADP, generating one ATP molecule in the process.

There is are 2 molecules of pyruvate produced and a net gain of 2 ATP per molecule of glucose commited to the glycolytic pathway. 3. Distinguish between the aerobic and anaerobic metabolism of glucose with reference to the enzymes involved and the comparative efficiencies of each pathway with respect to ATP generation.

Substrate Level Phosphorylation •

Substrate-level phosphorylation can be defined as the production of ATP by the direct transfer of a high-energy phosphate group from an intermediate substrate in a biochemical pathway to ADP, such as occurs in glycolysis. phosphoglycerate kinase

e.g. 1,3-bisphosphoglycerate

3-phosphoglycerate ADP

ATP

This is in contrast to oxidative phosphorylation, where ATP is produced using energy derived from the transfer of electrons in an electron transport system

Pyruvate has Two Possible Fates in Anaerobic Conditions •

Alcoholic fermentation:

.

pyruvate decarboxylase

pyruvate

acetaldehyde H+

CO2

alcohol dehydrogenase

acetaldehyde

ethanol

NADH + H+ •

NAD+

this is characteristic of yeasts and can occur under anaerobic conditions

Generation of lactate:

lactate dehydrogenase

17


pyruvate

lactate NADH + H+

-

NAD+

this is also anaerobic and is characteristic of mammalian muscle during intense activity when oxygen is a limiting factor

Regeneration of NAD+ • •

Both alcoholic fermentation and the generation of lactate serve one common purpose: They allow NAD+ to be regenerated and thus glycolysis to continue, in conditions of oxygen deprivation. i.e. conditions in which the rate of NADH formation by glycolysis is greater than its rate of oxidation by the respiratory chain.

Anaerobic vs Aerobic Metabolism • •

From the anaerobic metabolism of glucose we only generate 2 molecules of pyruvate and 2ATP molecules (net). This contrasts poorly to the complete oxidative phosphorylation of glucose which can yield 38 molecules of ATP. 4. Describe the reactions catalysed by lactate dehydrogenase and creatine kinase and explain the diagnostic relevance of their appearance in plasma.

Lactate Dehydrogenase as a Diagnostic Tool • • •

Lactate dehydrogenase catalyses the inter-conversion of pyruvate and lactate. LDH is present in many body tissues, especially the heart, liver, kidney, skeletal muscle, brain blood cells and lungs. Elevated levels can be used to diagnose several disorders including: -

stroke heart attack liver disease (eg. hepatitis) muscle injury muscular dystrophy pulmonary infarction

Creatine Kinase as a Diagnostic Tool • In muscle, the amount of ATP needed during exercise is only enough to sustain contraction for around one second. • Thankfully a large reservoir of creatine phosphate is on hand to buffer demands for phosphate (25mM creatine phosphate c.f. 4mM ATP in resting muscle). • DG (hydrolysis) = -31 kJ/mole (ATP) & -43.1 kJ/mole (CP) Creatine kinase

creatine phosphate

creatine +ATP 18


ADP + H+ • •

When a muscle is damaged, creatine kinase leaks into the bloodstream. Either total levels of creatine kinase or the tissue specific isoform can be measured to help to determine which tissue has been damaged. Elevated levels can be used to: -

ATP

diagnose myocardial infarction (heart attack) determine the extent of muscular disease evaluate the cause of chest pain help discover the carriers of muscular dystrophy (Duchenne)

The total creatine kinase test is about 70% accurate whilst isoenzyme testing is about 90% accurate. 5. Outline the oxidative decarboxylation reaction catalysed by pyruvate dehydrogenase, with reference to the product and the five co-enzymes required by this enzyme complex.

Generation of Acetyl CoA Pyruvate dehydrogenase complex

pyruvate + HS-CoA

acetyl CoA + CO2 NAD+

NADH

• •

This series of reactions occurs in the mitochondria of the cell. The acetyl CoA thus formed is committed to entry into the citric acid cycle and can ultimately produce ATP by the process of oxidative phosphorylation • This is the committed step for entry of pyruvate into the TCA cycle although in reality there is a lot more happening. • The pyruvate dehydrogenase complex is gigantic (in molecular terms) and not only consists of three individual enzymes but also five co-factors: Enzyme Prosthetic Group pyruvate decarboxylase (E1) thiamine pyrophosphate (TPP) lipoamide reductase-transacetylase (E2) lipoamide dihydrolipoyl dehydrogenase (E3) FAD (Flavine Adenine Dinucleotide) • Prosthetic groups such as lipoamide are a permanent part of the complex, whereas NAD+ and CoA bind reversibly to enzymes. Thiamine Pyrophosphate (TPP) • Derivative of the B1 vitamin (thyamine) • Readily loses a proton and the resulting carbanion attacks that of pyruvate to yield hydroxyethyl-TPP. • A deficiency of thiamine (vitamin B1) is the cause of Beri-Beri , whose symptoms include damage to the peripheral nervous system, weakness of the musculature and decreased cardiac output. The brain is particularly vulnerable as it relies heavily on glucose metabolism. 19


Lipoamide • •

The long flexible arm of the molecule allows the dithiol group to swing from one active site to another within the complex. Arsenite (AsO33-) and mercury have a high affinity for neighbouring sulphydryl groups, such as those that occur in reduced lipoamide and will readily inhibit pyruvate dehydrogenase.

Flavine Adenine Dinucleotide •

FAD accepts and donates 2 electrons with 2 protons (2 H):

FAD + 2 e- + 2 H+

FADH2

The Pyruvate Dehydrogenase Complex

• • • • •

• •

Decarboxylation of pyruvate to give hydroxyethyl TPP. Oxidation & transfer to lipoamide to give acetylipoamide. Transfer of the acetyl group to CoA to give acetyl CoA. Regeneration of oxidised lipoamide. Regeneration of oxidised FAD, generating NADH. 6. Describe the different processes by which the fatty acid palmitate and the amino acid alanine are converted into acetyl-CoA. Acetyl CoA is produced from both types of major food molecules within the mitochondria of cells. Thus it is the location where most of the cell's oxidation reactions occur and also where the majority of the cell’s ATP is made.

20


Fatty Acid Metabolism •

By virtue of being fully reduced (i.e carbon skeletons 'saturated' with hydrogens) the oxidation of fatty acids constitutes the most compact fuel for the body's energy requirements and as a result, fatty acid oxidation yields several times the usable chemical energy that carbohydrates can deliver. On a weight basis, the caloric yield from fatty acids is about double that from carbohydrates. More than half of the body's energy needs including the liver, but not the brain, comes from fatty acid oxidation and this is enhanced during fasting over long periods of time. Fatty Acids are metabolised in the mitochondria in several stages. Firstly, they are converted into an acyl CoA species:

Fatty acid + + ATP + HS i.e. ATP •

• • •

Acyl CoA + AMP + PPi

AMP, 2 high energy bonds are used.

The acyl coA species then undergoes a sequence of dehydration, hydration, oxidation and thiolysis reactions (collectively called b-oxidation) resulting in production of one molecule of acetyl CoA and an acyl CoA species which is 2 carbons shorter than the original.

Eg. Palmitic acid (16C) •

CoA

myristyl-CoA (14C) + acetyl CoA (2C)

The β-oxidation reactions continue to consecutively remove 2-carbon units from the acyl CoA therby producing acetyl CoA. On the final cycle (4-carbon fatty acyl CoA intermediate), two acetyl CoA molecules are formed. From just 7 b-oxidation reactions, the 16-carbon palmitoyl CoA molecule produces 8 molecules of acetyl CoA. During each cycle one molecule each of FADH2 and NADH are produced. The overall reaction of β-oxidation of palmitoyl CoA is:

palmitoyl CoA + 7 FAD + 7 NAD+ + 7 H2O + 7 CoA 8 acetyl CoA + 7 FADH2 + 7 NADH Amino Acid Metabolism •

• • •

Amino acid metabolism can be 'separated' into pathways depending on the number of carbon atoms the amino acid possesses. All of the degradation pathways produce common end products which can enter the TCA. The majority of the degradation takes place in the liver. C3 family e.g. alanine, serine (glycine), and cysteine are all degraded to pyruvate. C4 family e.g. aspartate and asparagine are degraded to oxaloacetate. C5 family e.g. glutamine, proline, arginine, and histidine, all of which are converted to a-ketoglutarate.

Protein Metabolism

21


Alanine (C3) undergoes transamination by the action of the enzyme alanine aminotransferase.

Pyruvate can enter the TCA cycle, while glutamate is re-converted to aketoglutarate by glutamate dehydrogenase, generating NH4+ which is ultimately converted to urea. Persistently elevated levels of alanine aminotransferase are a diagnostic for hepatic disorders such as Hepatitis C.

Summary • •

Glycolysis is central to metabolism in mammals, producing 2 moles of ATP for every mole of glucose and it relies upon the formation of a high energy compound which is subsequently split to liberate energy. Under aerobic conditions, pyruvate produced by glycolysis can be dehydrogenated by the actions of a giant multimeric enzyme, PDH, to generate acetyl CoA, a substrate for the TCA cycle in mitochondria and a prelude to oxidative phosphorylation. Both amino acids and fatty acids can be oxidised to generate metabollically useful components such as pyruvate and acetyl CoA.

22


Metabolism 4 -Metabolic Pathways and ATP Production II Anil Chopra 1. Outline the Krebs or TCA (tricarboxylic acid cycle) with particular reference to the steps involved in the oxidation of acetyl Co-A and the formation of NADH and FADH2 and the cellular location of these reactions.

The Three stages of Cellular Metabolism cytosol

Proteins Amino Acids

Glucose

NH2

TCA cycle

ATP

Pyruvate

NADH

Acetyl CoA

Oxidative Phosphoryation

NADH

ATP

H20

O2

Glycolysis

mitochondrion

Simple sugars

CO2 Polysaccharides Fatty Acids &Glycerol

Fats

The Manufacture of Acetyl Co Enzyme A • Entry to the TCA cycle via conversion into acetyl CoA:

• • •

The thioester bond is a high-energy linkage, so it is readily hydrolysed, enabling acetyl CoA to donate the acetate (2C) to other molecules. RNA ancestry suggests it is of primeval origin. The pyruvate dehydrogenase complex: pyruvate dehydrogenase complex

Pyruvate + HS-CoA

acetyl CoA + CO2

23


NAD+ NADH

1. Decarboxylation of pyruvate to give hydroxyethyl TPP. 2. Oxidation & transfer to lipoamide to give acetylipoamide. 3. Transfer of the acetyl group to CoA to give acetyl CoA. 4. Regeneration of oxidised lipoamide. 5. Regeneration of oxidised FAD, generating NADH. • • • • •

Acetyl CoA can also be manufactured from palmitic acid (a fatty acid). The β-oxidation reactions continue to consecutively remove 2-carbon units from the acyl CoA therby producing acetyl CoA. On the final cycle (4-carbon fatty acyl CoA intermediate), two acetyl CoA molecules are formed. From just 7 b-oxidation reactions, the 16-carbon palmitoyl CoA molecule produces 8 molecules of acetyl CoA. During each cycle one molecule each of FADH2 and NADH are produced. The overall reaction of β-oxidation of palmitoyl CoA is: palmitoyl CoA + 7 FAD + 7 NAD+ + 7 H2O + 7 CoA 8 acetyl CoA + 7 FADH2 + 7 NADH

The TCA Cycle • • • •

a.k.a. The Tricarboxylic Acid (TCA) cycle or The Citric Acid Cycle. A continuous cycle of eight reactions, starting with 2 carbon atoms from acetyl CoA being condensed with the 4 carbon unit of oxaloacetate to give a 6 carbon unit , citrate. The thio-ester linkage of the acetyl CoA allows it to be readily donated to oxaloacetate. Each turn of the cycle produces two molecules of CO2 (waste) plus three molecules of NADH, one molecule of GTP and one molecule of FADH2.

24


citrate synthase

Step 1: oxaloacetate (4C)

citrate (6C) HS-CoA + H+

Acetyl CoA (2C)

- transfer to the oxaloacetate of 2C from acetyl CoA •

Step 2:

aconitase

citrate (6C)

isocitrate (6C)

- isomerisation of citrate to give isocitrate •

Step 3:

isocitrate dehydrogenase

isocitrate (4C)

α-ketogluterate (5C) NAD+

NADH + H+ + CO2

oxidation of isocitrate to give α-ketoglutarate dehydrogenation and decarboxylation α-ketoglutarate dehydrogenase complex

Step 4:

α-ketoglutarate (5C) + HS-CoA

succinyl-CoA

(4C) NAD+ •

similar to reaction catalysed by PDH dehydrogenation and decarboxylation succinyl CoA synthetase

Step 5: succinyl CoA (4C)

H2O + GDP + Pi -

NADH + H+ + CO2

succinate (4C) + HS-CoA GTP (guanisone triphosphate)

CoA is displaced by a phosphate molecule which is subsequently transferred to GTP only stage that directly forms GTP / ATP (in bacteria and plants) GTP itself can act as a phophoryl donor in protein synthesis or signal transduction processes Alternatively a phosphate grou can be transferred to that of ADP to generate ATP – catalysed by nucleoside diphosphokinase 25


Step 6:

succinate dehydrogenase

succinate (4C)

fumarate (4C) FAD

FADH2

oxidation of succinate generating some FADH2 dehydrogenation fumerase

Step 7: fumerate (4C)

malate (4C) H2O

addition of a water molecule – breaking a double bond malate dehydrogenase

Step 8: malate (4C)

oxaloacetate (4C) NAD+

-

NADH + H+

the last step – dehydrogenation of malate to give oxaloacetate, the starting point of the cycle

Location of the TCA Cycle Enzymes •

• •

The Krebs cycle enzymes are soluble proteins located in the mitochondrial matrix space, except for succinate dehydrogenase, which is an integral membrane protein that is firmly attached to the inner surface of the inner mitochondrial membrane. Here, it can communicate directly with components in the respiratory chain, as we shall see in the next lecture. The majority of the energy that derives from the metabolism of food is generated when the reduced coenzymes are re-oxidised by the respiratory chain in the mitochondrial inner membrane in a process known as oxidative phosphorylation. The Krebs cycle only operates under aerobic conditions, as the NAD+ and FAD needed are only regenerated via the transfer of electrons to O2 during oxidative phosphorylation.

Importance of the TCA Cycle • •

ATP production by glycolysis and the Krebs cycle is only a prelude to oxidative phosphorylation. The function of the Krebs cycle is to produce the reduced co-factors NADH and FADH2 which are re-oxidised during osidative phosphorylation to yield the following:

26


three ATP molecules formed by the re-oxidation of each NADH molecule two ATP molecules formed by the re-oxidation of each FADH2 molecule.

Therefore from the Krebs cycle – oxidation of 1x acetyl coA gives: -

3x NADH + 1x FADH2 + 1x GTP = 12x ATP

2. Outline the glycerol phosphate shuttle and the malate-aspartate shuttle, in particular stating why these mechanisms are required and understand the concept of transamination with reference to the malate-aspartate shuttle. • • •

NADH produced in glycolysis needs to enter the mitochondria to be utilised by the process of oxidative phosphorylation and to regenerate NAD+. Remember, there is only a finite amount of NAD+ and unless it is regenerated, glycolysis will very quickly grind to a halt. NADH, or more accurately, its high-energy electrons, crosses from the cytosol into the matrix of the mitochondria by two methods: -

the glycerol phosphate shuttle – skeletal muscle, brain the malate- aspartate shuttle – liver, kidney, heart

The Glycerol Phosphate Shuttle • •

Electrons from NADH, rather than NADH itself are carried across the mitochondrial membrane via the carrier glycerol-3-phosphate. A cytosolic glycerol dehydrogenase (G-DH) transfers electrons from NADH to glycerol 3phosphate, which can diffuse into the mitochondria. There, a membrane bound form of the same enzyme transfers them to FAD.

The Malate-Aspartate Shuffle • •

This takes place primarily in the heart and liver and uses two membrane carriers and four enzymes. The net reaction in terms of NADH is:

NADHcytoplasmic + NAD+mitochondrial • •

NAD+cytoplasmic + NADHmitochondrial

Hydrogen is transferred from cytoplasmic NADH to oxaloacetate to give malate, a reaction catalysed by cytosolic malate dehydrogenase (MDH). Malate can be transported into the mitochondria where it is rapidly re-oxidised by NAD+ to give oxaloacetate and NADH (catalysed by mitochondrial MDH). 27


• • •

However, oxaloacetate has now been being depleted from the cytoplasm and is accumulating within the mitochondrial matrix. Since it cannot cross the matrix membrane, the problem cannot be solved by by simply transferring oxaloacetate back to the cytoplasm. Instead, the cell uses a transamination reaction to take an amino group from glutamate and transfer it to oxaloacetate, giving aspartate. This aspartate then crosses the matrix membrane, via an amino acid transporter, and is duly converted by the same transamination reaction in reverse, back to oxaloacetate. Transamination is therefore pivotal to the malate-aspartate shuttle – it is defined as a reaction in which an amine group is transferred from one amino acid to a keto acid, thereby forming a new pair of amino and keto acids. A more accurate diagram of the malate-aspartate shuffle is therefore:

A B

• •

A = alpha-ketoglutarate transporter – exchanges alpha-ketoglutarate for malate B = glutamate/aspartate transporter – exchanges glutamate for aspartate

3. Explain in general terms the relationship between TCA intermediates and those pathways involved in amino acids synthesis and breakdown. • •

The general strategy of amino acid degradation is to remove the amino group (which is eventually excreted as urea) whilst the carbon skeleton is either funnelled into the production of glucose or fed into the Krebs cycle Degradation of all twenty amino acids gives rise to only seven molecules, pyruvate, acetyl CoA, acetoacetyl CoA, a-ketoglutarate, succinyl CoA, fumarate and oxaloacetate. 28


4. Calculate the total yield of ATP obtained from the complete oxidation of one glucose molecule. glycolysis

glucose

2x pyruvate (net 2x ATP + 2x NADH) PDH

2x pyruvate + CoA + NAD+

2x acetyl CoA + 2x NADH

TCA cycle

2x acetyl CoA

6x NADH + 2x FADH2 + 2x GTP

This gives 2x ATP + 10x NADH (3x ATP each) + 2x FADH2 (2x ATP each) + 2x GTP = 38x ATP from one molecule of glucose 5. Compare the complete oxidation of one glucose molecule, with the beta oxidation of palmitic acid with reference to the ATP produced per molecule of each substrate. 1st step

palmitate + ATP + HS-CoA (loss 2xATP)

palmitoyl CoA + AMP + PPi

β-oxidation

palmitoyl CoA + 7x FAD + 7x NAD+ + 7H2O + 7x CoA CoA + 7x FADH2 + 7x NADH

8x acetyl

TCA cycle

8x acetyl CoA

24x NADH + 8x FADH2 + 8x GTP

This gives 31xNADH (3x ATP each) + 15x FADH2 (2xATP each) + 8x GTP – 2xATP = 129x ATP from one molecule of plamitate – about 5 times that from glucose 6. Give two examples of the use of NADPH in reductive biosynthesis. • •

Glycolysis and the Krebs cycle provide the starting point for many biosynthetic reactions. The amino acids, nucleotides, lipids, sugars, and other molecules shown here as products, in turn, become the precursors for the many of the macromolecules of the cell.

29


• • •

Each black arrow in this diagram denotes a single enzyme-catalysed reaction. Red arrows generally represent multi-step pathways. If Krebs cycle intermediates are drawn off for biosynthesis then they must be replenished, otherwise the cycle will grind to a halt. e.g. If oxaloacetate is removed, acetyl CoA cannot enter and glycolysis backs up. Thankfully some enzymes can catalyse anaplerotic reactions (from the Greek to fill up) which can regenerate Krebs cycle intermediates.

Nicotinamide Adenine Dinucleotide Phosphate (NADP+) • • • • • • •

This is a relative of NAD+ differing only by a phosphate group attached to one of the ribose rings NADP+ is also an electron carrier. Like NAD+, NADP+ can pick up two high energy electrons and in the process, a proton (H+) collectively known as a hydride ion (H-). It then forms NADPH. The phosphate group of NADP+ does not participate in electron transfer, but gives it a slightly different conformation, meaning that it will bind to different enzymes than NAD+. The hydride ion is held in a high-energy linkage, allowing it to be easily transferred to other molecules. NADPH takes part in anabolic reactions, whereas NADH takes place in catabolic reactions. The use of different co-factors for sets of reactions is a classical “division of labour”a common theme throughout biology. It allows electron transport in catabolism to be kept separate to that of anabolism.

NADPH is a Co-factor in the Biosynthesis of Cholesterol

• •

NADPH helps to catalyse the final reaction of several, that lead to cholesterol synthesis. The C=C bond is reduced by the transfer of a hydride ion (two electrons plus a proton from solution, H-).

NADPH is a Co-factor in the Biosynthesis of RNA

30


Metabolism 5 - Mitochondria and Oxidative Phosphorylation 1. Outline the proposed evolutionary origins of mitochondria. • • •

Mitochondria are believed to be the evolutionary descendants of a prokaryote that established an endosymbiotic relationship with the ancestors of eukaryotic cells. This is thought to have occurred early in the history of life on earth and that following this, many of the genes needed for mitochondrial function were moved (translocated) to the nuclear genome. More recently, the elucidation of the complete genome of Rickettsia prowazekii has revealed that several genes are closely related to those found today in mitochondria.

Support for the Theory: • • • • •

Mitochondria can only arise from pre-existing mitochondria and chloroplasts. Mitochondria possess their own genome and it resembles that of prokaryotes, being a single circular molecule of DNA, with no associated histones. Mitochondria have their own protein-synthesizing machinery, which again resembles that of prokaryotes not that of eukaryotes. The first amino acid of their transcripts is always fMet as it is in bacteria and not methionine (Met) that is the first amino acid in eukaryotic proteins). A number of antibiotics (e.g., streptomycin) that act by blocking protein synthesis in bacteria also block protein synthesis within mitochondria and chloroplasts. They do not interfere with protein synthesis in the cytoplasm of the eukaryotes.

2. Draw a cross sectional representation of a mitochondrion, and label its component parts.

• •

The reactions of oxidative phosphorylation take place on the inner membrane. The folds of the christae increase the surface area for these reactions to take place. 31


3. Describle the electron transport chain in mitochondria with reference to the functions of coenzyme Q (ubiquinone) and cyctochrome c. Two Steps of Oxidative Phosphorylation: • The translocation or movement of protons from within the matrix of the mitochondria – controlled by the electron transport chain. • The pumped protons are allowed back into the mitochondria through a specific channel, which is coupled to an enzyme which can synthesise ATP known as ATP synthase. The Electron Transport Chain • •

• • • • •

The electron transport chain is a chain of three complexes and two mobile carriers whih act as electron carriers. Membrane complexes - NADH dehydrogenase complex - cytochrome b-c1 complex - cytochrome oxidase complex Mobile carriers - ubiquinone (co-enzyme Q) - cytochrome C These proteins accept electrons and in doing so, a proton (H+) from the aqueous solution. As electrons pass through each of the complexes, protons are pumped to the intermembrane space. Each unit of the chain has a higher affinity for electrons than the previous unit, allowing them to flow in a logical order. The transfer of electrons from one complex to another is energetically favourable and so the electrons lose energy as they progress along the chain.

Ubiquinone • • • •

Ubiquinone can pick up either one or two electrons (with an H+ from solution) and pass them to cytochrome b-c1 complex. Its hydrophobic tail confines it to the lipid bilayer of the membrane where it is needed. It is the entry point for electrons donated by FADH2 since succinate dehydrogenase can communicate directly with it. As fewer protons are pumped to the intermembrane space than for NADH, less ATP is produced.

Cytochrome Oxidase • •

Cytochrome oxidase is involved in the final electron transfer step. It receives 4 electrons from cytochrome c and passes them to oxygen to generate water: 4e- + 4H+ + O2

2H2O 32


In addition, 4 protons are pumped to the intermembrane space, enhancing the proton gradient.

Redox Reactions • • • •

The reactions that take place in the electron transport chain are redox reactions since a reduced substrate donates electrons and an oxidised substrate accepts them (thus electrons move along the transport chain). The ability of a redox couple to accept of donate electrons is known as the reduction potential, or redox potential. Standard redox potential is given the symbol E’0. Positive E’0 value shows redox couple has a tendency to donate electrons and vice versa.

4. Outline the chemiosmotic theory. Two Steps of Oxidative Phosphorylation: • •

The translocation or movement of protons from within the matrix of the mitochondria – controlled by the electron transport chain. The pumped protons are allowed back into the mitochondria through a specific channel, which is coupled to an enzyme which can synthesise ATP known as ATP synthase.

Chemiosmosis: • • •

The proton motive force that drives H+ back into the matrix space consists of a pH gradient and a transmembrane electrical potential. This flow of protons back into the matrix is coupled to ATP synthesis. Protons flow back into the matrix through the ATP synthase molecule, which produces ATP from ADP + Pi.

ATP Synthase: • • • • •

ATP synthase is a multimeric enzyme consisting of a membrane bound part (F0) and a part which projects into the matrix space (F1). Each part consists of three different sub units. When hydrogen ions flow through the membrane via a pore, the disc of c subunits is compelled to rotate. The γ-subunit in the F1 unit is fixed to the disc and therefore rotates with it. However, the α and β subunits in the F1 unit cannot rotate because they are locked in a fixed position by the b subunit, which is anchored to subunit a in the membrane.

33


• • • •

As the γ subunit functions as an asymmetrical axle, the β subunits are compelled to undergo structural changes. This rotation drives the transitions of the catalytic portions of the β subunits, which in turn, alters their affinities for ATP and ADP. As a consequence, torsional energy flows from the catalytic subunit into the bound ADP and Pi to promote the formation of ATP. The direction of proton flow dictates ATP synthesis vs ATP hydrolysis.

5. Explain why carbon monoxide, cyanide, malonate and oliogomycin are poisonous in terms of their effects on specific componenets of the electron transport chain. ATP Consumption and Cell Death • Human body synthesises about 70kg of ATP a day. • Each ATP molecule has a life span of 1-5 mins. • Any interruption of oxidative phosphorylation and therefore ATP synthesis means that the cell rapidly becomes depleted of ATP and is likely to die. • Most common cause of this is lack of oxygen – hypoxia (diminished) or apoxia (total). • Death of cell may be within a few minutes (neurons) or a few hours (muscles). Cyanide and Carbon Monoxide Poisoning • Classified as supertoxic – a few drops ingested can kill. • Cyanide (CN-) and azide (N3-) bind with high affinity to the ferric (Fe3+) form of the haem group in the cytochrome oxidase complex. • This blocks the flow of electrons through electron transport chain and consequently the production of ATP. • In a similar way, CO binds to the ferrous (Fe2+) form of the haem group, also blocking the flow of electrons. Malonate Poisoning • Malonate closely resembles succinate and acts as a competitive inhibitor of succinate dehydrogenase. • This Kreb’s Cycle enzyme resides in the the inner membrance and passes FADH2 directly to ubiquinone. • Malonate therefore slows down the flow of electrons from succinate to ubiquinone, slowing down ATP production. Oliogomycin Poisoning • Oliogomycin is an antibiotic that inhibits oxidative phosphorylation by binding within the ‘stalk’ of ATP synthase • This blocks the flow of protons through the enzyme. • ATP synthesis is inhibited and a backlog of protons builds up in the intermembrane space. • This inhibits the flow of electrons through the electron transport chain as the H+ concentration in the intermembrane space reaches saturation point – no more can be pumped out.

34


6. Describe how oxidative phosphorylation can be measured experimentally. The Oxygen Electrode • The oxygen electrode measures the concentration of oxygen in a solution contained in the chamber of the apparatus. • The base of the chamber is formed by a Teflon membrane permeable to oxygen. Underneath this membrane is a compartment containing two electrodes – a platinum cathode and a silver anode. • A small voltage is applied between the silver anode and the platinum cathode. • Oxygen diffuses through the Teflon membrane and is reduced to water at the cathode. • The circuit is completed at the anode, which is slowly corroded by the KCl electrolyte. • The resulting current is proportional to the oxygen concentration in the sample chamber. Measuring Oxidative Phosphorylation • A suspension of mitochondria from homogenised tissue are incubated within a sealed incubation chamber containing and isotonic medium containing substrate eg succinate and Pi. • The addition of ADP causes a sudden burst of oxygen uptake as the ADP s converted into ATP – this is termed coupled respiration • By adding various substances to the camber we can determine their effects on oxidative phosphorylation. Summary • • •

• •

Mitochondria are present in almost all eukaryotic cells and are believed to be evolutionary descendants of an earlier prokaryote life form. The majority of ATP produced within the body occurs a result of oxidative phosphorylation, a process which takes place within the mitochondria. In this process, electrons from the reduced co-enzymes NADH and FADH2 are passed via a series of enzyme complexes, called the electron transport chain, which ultimately results in the reduction of oxygen to water and the pumping of protons out of the mitochondrial matrix, to generate a proton gradient. These protons re-enter the matrix via the molecular turbine ATP synthase, which couples the resultant kinetic energy to ATP production. Compounds which inhibit either the electron transport chain or the proton gradient, disrupt oxidative phosphorylation which be measured experimentally with an oxygen electrode.

35


Metabolism 6 - Lipids & Membranes Objectives: • Describe the structure of: fatty acids, triglycerides, phospholipids, cholesterol and sphingomyelin. • Give examples of how the lipid composition can differ for different cellular membranes, and indicate the significance of this. • Outline the pathway for synthesis of fatty acids. • Distinguish between the pathways for synthesis and metabolism of fatty acids in terms of: substrates and products, coenzymes used, carrier molecules and cellular location. ● Describe the structure of: fatty acids, triglycerides, phospholipids, cholesterol and sphingomyelin: • Fatty acids: Fatty acids are the simplest of all lipids and are constituents of more complex lipids. They have hydrophilic (“water-liking”) heads, and hydrophobic (“water-disliking”) tails. The fatty acid chain can either be composed of saturated bonds (as is the case with Stearic acid, shown in diagram). The acid chain can also contain unsaturated bonds (as is the case with Oleic acid, also shown in the diagram). • Triglycerides: As the name suggests, triglycerides contain three fatty acids attached to a glycerol backbone. Their main function is that of an energy store. 36


R1, R2 and R3 indicate the three fatty acids. • Phospholipids: Phospholipids are essentially the same as triglycerides, but one of the fatty acids has been replaced by a phosphate group. The phosphate group is very hydrophilic, and the fatty acid tails are as always hydrophobic. This gives rise to the formation of phospholipids bilayers – important in the structure of cell membranes. • Cholesterol:

The structure of cholesterol is given in more detail in the next lecture. The main point to note about its structure is that all cyclohexane rings are in the chair conformation; this gives cholesterol a planar, rigid structure.

• Sphingomyelin: This composition

is

the of

37


sphingomyelin. As can be seen it has two components; phosphocholine and ceramide. Sphingomyelin is a common membrane lipid.

● Give examples of how the lipid composition can differ for different cellular membranes, and indicate the significance of this: • The cell or plasma membrane is best described as a 2-D fluid structure or orientated proteins and lipids. It is a continuous double layer (bilayer) about 5nm thick. The lipids and proteins in the membrane are held non-covalently, and the proteins the bilayer contains may be either integral (part of the membrane) or peripheral (on the outside of the membrane)…

• The degree of fluidity of the cell membrane (the ease with which its lipid molecules can move about) is important for membrane function, and thus must be maintained within certain limits. 38


• The fluidity of a lipid bilayer at a given temperature depends on its phospholipids composition, and especially on the nature of the hydrocarbon tails: the closer and more regular the packing of the tails, the more viscous the and less fluid the bilayer will be. There are two major properties of hydrocarbon tails that affect their packing in the bilayer –one is their length, and the other is their unsaturation (that is the number of double bonds they contain). • Hydrocarbon tails of phospholipids vary in length between 14 and 20 carbon atoms, 18-20 being the most usual. A shorter chain reduces the tendency of the hydrocarbon tails to interact with one another, therefore increasing the fluidity of the membrane. • Each double bond in a hydrocarbon tail creates a kink in the tail, making it harder for the tails to pack closely together. Thus, lipid bilayers that contain a large proportion of unsaturated hydrocarbon tails are more fluid than those with lower proportions. • Membrane fluidity is important in cells for many reasons. It (the cell membrane) enables membrane proteins to diffuse rapidly in the plane of the bilayer and to interact with one another (which is crucial in cell signalling for example). It also provides a simple means of distributing membrane lipids and proteins by diffusion from sites where they are inserted into the bilayer (after their synthesis) to other regions of the cell. It allows membranes to fuse with one another and mix their molecules, and it ensures membrane molecules are evenly distributed between daughter cells when a cell divides. • In animal cells, membrane fluidity is regulated by the sterol cholesterol (which is absent in yeasts, plants and bacteria). These short, rigid molecules are present in especially large amounts in the plasma membrane where they fill the space between neighbouring phospholipid molecules that are caused by kinks in unsaturated fatty acids. In this way, cholesterol has the ability to stiffen the bilayer making it less fluid and permeable. ● Outline the pathway for synthesis of fatty acids (Lipogenesis):

39


AcetylCoA is the key

intermediate between fat and carbohydrate metabolism.

• Essentially, the overall reaction occurring is:

But there are many steps in between… • 1st STEP: PRODUCTION OF MALONYL CoA (catalysed by the enzyme Acetyl CoA carboxylase):

40


• 2nd STEP: ACTIVATION BY ACYL CARRIER PROTEIN (similar to CoA activation in β-oxidation). • 3rd STEP: ELONGATION BY SUCCESSIVE ADDITION OF 2-CARBON UNITS (this is catalysed by the enzyme FA Synthase):

Note how the oxidative degradation (all of the reactions on the left-hand side of the diagram (pink)) is very similar to the synthesis (all of the reactions on the right-hand side of the diagram (blue)) of a fatty acid.

41


• Fatty acid synthase – mechanism of reaction…

42


• The overall reaction is given as…

• Palmitate can undergo further metabolism: 1. Esterification to form triacylglycerols. 2. Formation of other fatty acids, unsaturated and longer chains… Desaturation, elongation, formation of monounsaturated FA’s - catalysed in different cellular compartments. • Lipogenesis is regulated:

1. Feedback inhibition of palmitoyl CoA to: A) AcetylCoA carboxylase. B) FA synthase. C) Pentose phosphate pathway. 2. Acetyl CoA carboxylase regulation by hormones.

43


3. Transcriptional regulation of acetyl CoA carboxylase and FA synthase (activated by insulin and inhibited by glucagon. • Metabolism of fatty acids: The pathway for the metabolism of fats is given below (the actual pathway is highlighted in the pale yellow colour)‌

44


Metabolism 7 - Cholesterol Objectives: • Explain the physiological functions of cholesterol in membrane stability. • Outline the synthesis of cholesterol from acetate. • Outline the synthesis of bile acids and steroid hormones from cholesterol. • Describe the mechanism of transport of cholesterol around the body and its uptake into cells. • Draw a diagram of low density lipoprotein (LDL). • Explain why disturbances in cholesterol homeostasis cause disease. • Give an example of how a selective enzyme inhibitor can be used as a pharmacological agent in controlling cholesterol metabolism. ● Explain the physiological functions of cholesterol in membrane stability: • In animal cells, membrane fluidity is regulated by the sterol cholesterol (which is absent in yeasts, plants and bacteria). These short, rigid molecules are present in especially large amounts in the plasma membrane where they fill the space between neighbouring phospholipid molecules that are caused by kinks in unsaturated fatty acids. In this way, cholesterol has the ability to stiffen the bilayer making it less fluid and permeable. ● Outline the synthesis of cholesterol from acetate: • Step 1: Formation of Mevalonate…

See next page…

45


1.1 HMGCoA Reductase as the regulated step in cholesterol synthesis

1.2

1.3

• Step 2: Mevalonate to Squalene (Isoprenoid metabolism): Again, there are 3 stages to this step of the synthesis of cholesterol: 1) Mevalonate to C5 units (isoprene units) 2) Head-to-tail condensations of isoprene units 3) Branched pathway at farnesyl pyrophosphate see next page‌ 46


47


• Step 3: Squalene to Cholesterol:

C5 C10 C15

C30

48


● Outline the synthesis of bile acids and steroid hormones from cholesterol: • The synthesis of Steroid Hormones…

49


• Levels of steroid hormone controlled by the rate of synthesis. • Cholesterol desmolase generates pregnenolone the precursor of all steroid hormones. • There are 5 classes of steroid hormone: 1. Progestins (progesterone, 17-Hydroxypregnenolone, 17Hydroxyprogesterone) 2. Glucocorticoids 3. Mineralocorticoids 4. Androgens 5. Estrogens • The synthesis of Bile Acids…

Conjugated bile acids 50


• This pathway represents the major route for elimination of cholesterol via the GI tract. ● Describe the mechanism of transport of cholesterol around the body and its uptake into cells: • Dietary cholesterol is transported from the gut to the liver by chylomicrons. • Cholesterol is transported from the liver to the tissues by the low density lipoproteins (LDLs); these may be taken up by LDL receptors on cells; the LDL receptors are associated in clathrin coated pits, and so are frequently endocytosed; if LDL is bound, this is internalised also. Lysosomes then break up the vesicle contents into free cholesterol and amino acids (from the LDL apoproteins). • HDLs transport cholesterol from the tissues back the liver or to endocrine glands for steroid synthesis.

51


● Draw a diagram of low density lipoprotein (LDL):

Lipoproteins are used to transport hydrophobic triglycerides in the bloodstream. The shell’s outside is hydrophilic, and contains phospholipids, cholesterol and proteins like a normal cell membrane, and thus its inside is hydrophobic and suitable for fats; the different types of lipoprotein are classified by their density, which is high with high protein content and low with high fat content (i.e. high density lipoproteins (HDLs) have more protein:fat). ● Explain why disturbances in cholesterol homeostasis cause disease: In familial hypercholesterolaemia (FH) for example, there is a genetic defect that causes either an absence of or a mutation of the LDL receptor (as they do not migrate to clathrin coated pits and so are not endocytosed); this results in raised cholesterol levels in the blood, leading to a predisposition to atherosclerosis. Accelerated atherosclerosis means that sufferers have high early MI (myocardial infarction) risk. FH heterozygotes may be asymptomatic until their 40s, but homozygotes if untreated die in their teens (due to a complete absence of LDL receptors). ● Give an example of how a selective enzyme inhibitor can be used as a pharmacological agent in controlling cholesterol metabolism: • HMG-CoA reductase inhibitors such as Mevastatin inhibit cellular cholesterol synthesis.

52


Metabolism 8 - Membrane Trafficking 1. Explain the terms ‘endocytosis’ and ‘exocytosis’. • Exocytosis is the process of secreting macromolecular material from a cell. • It involves the fusion of a membrane-enclosed intracellular vesicle with the plasma membrance, followed by the opening of the vesicle and the emptying of its contenets to the outside. • Endocytosis is the mechanism of uptake of macromolecular material into a cell from the outside. • It typically involves formation of a coated pit on the plasma membrane, which buds off into the cytoplasm to form a coated vesicle which delivers its contents to an endosome. 2. Describe the pathway and cellular locations for synthesis, post-translational modification and exocytosis of a secreted protein. Secretory or Exocytic Pathway Endoplasmic reticulum plasma membrane

golgi apparatus (cis – medial

– trans)

At the Endoplasmic Reticulum • • • •

• • •

Protein synthesis occurs at the ribosomes. These are initially free in they cytosol, a common pool of ribosomes is used to synthesise both those proteins that remain in the cytosol and those that are transported into the ER. If a protein is destined for the ER, the ER signal peptide (first piece of the polypeptide to be synthesised) directs the ngaged ribosome to the ER membrane. The ribsosmes are then recycled after each round of protein synthesis. The ER is also the site of some post-translational modifications: - formation of disulfide bonds - folding - glycosylation (makes proteins stronger and more resistant to agressors) - specific proteolytic cleavages - assembly of multimeric proteins - tertiary structure constructed by ER membranes There is a quality control mechanism, as only proteins properly folded and glycolated can pass through ER exit. Unassembled of misfolded proteins are retaine in the ER and exported back to the cytosol where they are degraded. The proteins are then transported in vesicles to the golgi apparatus.

At the Golgi Apparatus • • •

More post-translational modifications take place at the golgi apparatus. At the cis golgi network, the phosphorylation of lysosomal proteins occurs. in the golgi stack, Man is removed and GlcNAc and Gal are added. 53


• •

At the trans golgi network NANA is added and the proteins are sorted. The golgi apparatus also returns ER resident proteins – recognised by KOEL receptors.

Sorting at the Trans Golgi Apparatus and the Plasma Membrane • • • • •

Here proteins are sorted and packaged into vesicle depending on whether they are to be released by constitutive or regulated secretion. Lysosomal emnzymes are also sorted from others. The mannose in these proteins is phosphorylated in the golgi stack producing mannose-6-phosphate. This is then recognised by a M6P receptor and these proteins are taken in a receptor dependent transport vesicle to a lysosome. The M6P receptors are recycled by budding from a late endosome.

3. Distinguish ‘constitutive’ and ‘regulated’ secretion. • • • •

Constitutive secretion is the secretion of plasma membrane lipids and soluble proteins. It is unregulated and so does not require any signals to generate the secretion. Regulated secretion requires an external signal from a hormone or neurotransmitter substance before it occurs. It is used from the secretion of proteins and other substances.

4. Describe the process of receptor-mediated endocytosis and the roles played by endocytic vesicles, early endosomes, late endosomes and lysosomes. • • • •

There are three fates for endocytosed material. Recycling involves substances entering the cell via the apical plasma membrane, being transferred to an early endosome and being release back out of the apical plasma membrane. The second fate is degradation in which substances are transferred from the apical plasma membrane, via an early endosome, to a lysosome where they are destroyed. The third fate is transcytosis in which substances enter the cell via the apical plasma membrane and exit via the basolateral plasma membrane, passing through an early endosome.

Receptor Mediated Endocytosis •

Example of this is endocytosis of LDLs.

54


• • • •

The LDL binds with an LDL receptor at the plasma membrane and is taken into a coated vesicle via endocytosis. The vesicle is uncoated in order to fuse with an early endosome. The LDL receptors bud off the early endosome and it becomes a late endosome. With the introduction of hydrolytic enzymes a lysosome is formed and free cholesterol is released into the cytosol.

5. Give a general description of the molecular mechanisms of vesicular transport within cells. • • • •

At the donor membrane, the cargo is sorted and a vesicle forms and buds off the plasma membrane. The vesicle then moves through the cytosol – vesicles move along microtubules to find receptors. When the vesicle reaches specific receptor molecules on the receptor membrane recognition occurs between this and specific molecules on the vesicle, resulting in vesicle tethering or docking to the acceptor membrane. The vesicle membrane and acceptor membrane then fuse together, opening the vesicle and releasing the contenets into the lumen of the acceptor organelle.

6. Give examples of diseases resulting from defects in the secretory and endocytic pathways. • •

A disease that results from a defect in the secretory pathway is cystic fibrosis. This results from blocks at the exit of the ER due to misfolding of the proteins.

A disease that results from a defect in the endocytic pathway is familial hypercholesterolaemia. Mutations in the LDL receptor mean that receptor mediated endocytosis of LDLs cannot take place.

• •

Intracellular trafficking is involved in many other diseases including more than 75 genetic diseases of syndromes, cancer and infections. 55


Metabolism 9 – Integration of Metabolism 1. Distinguish the features of metabolic activity in the following tissues: liver, brain, muscle, adipose tissue. Liver • Plays central role in coordinating metabolism throughout the body. • Immediate recipient of nutrients absorbed at the intestines. • Wide repertoire of metabolic processes. • Highly metabolically active and can interconvert nutrient types. • Central role in maintaining blood glucose at 4.0-5.5 mM. • Storage organ (glycogen). • Central role in lipoprotein metabolism. Brain • Has continuous high ATP requirement, cannot utilise fats. • Requires continuous supply of glucose for metabolism. • Cannot metabolise fatty acids • Ketone bodies (β-hydroxy-butyrate) can partially substitute for glucose. • Too little glucose (hypo-glycaemia) causes faintness and coma. • Too much glucose (hyper-glycaemia) can cause irreversible damage. Muscle • Can have periods of very high ATP requirement during vigorous contraction. • During vigorous contraction ATP consumption is faster than supply by oxidative phosphorylation (O2 diffusion is limiting). • Energy stores of glycogen (→glucose-6-P for glycolysis) and creatine phosphate (→ATP). • Under anaerobic conditions pyruvate is converted to lactate or alanine which can leave muscle and reach the liver via the blood. Adipose tissue • Is a long-term storage site for fats. 2. Give four examples of extracellular hormones which act as metabolic regulators. Secreted by pancreatic islets: • Insulin secreted when glucose levels rise: stimulates uptake and use of glucose and storage as glycogen and fat. • Glucagon secreted when glucose levels fall: stimulates production of glucose by gluconeogenesis and breakdown of glycogen and fat. Secreted by the adrenal glands: • Adrenaline (American = epinephrine): strong and fast metabolic effects to mobilise glucose for “flight of fight”. • Glucocorticoids: steroid hormones which increase synthesis of metabolic enzymes concerned with glucose. 3. Describe the changes in metabolic activity while eating and while fasting. • On having a meal, blood glucose initially rises and is controlled by: - Increased secretion of insulin (and reduced glucagon) from islets. - Increased glucose uptake by liver - used for glycolysis and glycogen synthesis. Acetyl-CoA produced is used for fatty acid synthesis. 56


•

Increased glucose uptake and glycogen synthesis in muscle. Increased triglyceride synthesis in adipose tissue. Increased usage of metabolic intermediates throughout the body due to general stimulatory effect on synthesis and growth. After a meal blood glucose starts to fall and is controlled by: - Increased glucagon secretion (and reduced insulin) from islets. - Glucose production in liver resulting from gluconeogenesis and glycogen breakdown. - Utilisation of fatty acid breakdown as alternative substrate for ATP production. - [NB adrenaline has similar effects on liver, but also stimulates skeletal muscle towards glycogen breakdown and glycolysis, and adipose tissue towards fat lipolysis to provide other tissues with alternative substrate to glucose]

After prolonged fasting (longer than can be covered by glycogen reserves): - Glucagon/insulin ratio increases further. - Adipose tissue begins to hydrolyse triglyceride to provide fatty acids for metabolism. - TCA cycle intermediates are reduced in amount to provide substrate for gluconeogenesis. - Protein breakdown provides amino acid substrates for gluconeogenesis. - Ketone bodies are produced from fatty acids and amino acids in liver to substitute partially the brain’s requirement for glucose. 4. Describe in general terms the relationship to glucose metabolism of: lipid synthesis and breakdown, amino acid synthesis and breakdown, synthesis of other components of macromolecules.

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5. Describe the metabolic processes during vigorous muscular activity and explain why acidosis can result. • During vigorous contraction ATP consumption is faster than supply by oxidative phosphorylation (O2 diffusion is limiting). • Further ATP by interconversion from creatine phosphate. • Glycogen stores provide glucose for anaerobic metabolism only (glycolysis). • Pyruvate is converted to lactate or alanine - otherwise it would build up and the pathway would be inhibited by excess product. • Lactate/alanine pass into the blood and the liver uses them to replenish glucose by gluconeogenesis .

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Anil - Metabolism  

Anil - Metabolism

Anil - Metabolism  

Anil - Metabolism

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