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Lecture 1: Introduction to protein structure 1) Describe the three main groups into which amino acids may be placed on the basis of the chemical nature of their side chains. • Amino acids with non-polar (hydrophobic) side chains • amino acids with polar (hydrophilic) side chains; • amino acids with charged (hydrophilic) side chains. 2) Outline the reaction in which amino acids are joined together. • Peptides are formed by a condensation reaction. The –OH group of a carboxyl group of one amino acid and a hydrogen from the amino group of another amino acid are released in the form of H2O resulting in a peptide bond between the two adjacent amino acids. 3) Sketch a trimeric peptide, illustrating the amino terminus, carboxyl terminus and side chains.

4) Give examples of the post translation modifications of amino acids. • Post-translation enhances the capabilities of the protein. • The addition of hydroxyl groups (eg. proline to hydroxyproline in collagen fibres- the constituent of skin, cartilage, teeth, bone etc…), the addition of the hydroxyl group helps to stabilise the fibres (hence a defiency in vit. C- which catalyses the hydroxyl reaction leads to scurvy) • N-linked glycosylation of asparagine residues of proteins increases their solubility and protects against enzymatic degradation; • carboxylation of glutamate (addition of a –COO- group), to give y-carboxyglutamate, which is critical for proteins within the blood clotting cascade as it increases their calcium binding ability. 5) Define the terms primary structure, secondary structure, tertiary structure & quaternary structure with respect to proteins. • Primary structure – Simply the linear sequence of amino acids that make up the protein. • Secondary structure – Local structural motifs within a protein eg. α–helices and β–pleated sheets; their existence within a protein is dictated by the primary structure or amino acid sequence. • Tertiary structure - the arrangement of the secondary structure motifs into compact globular structures called domains. • Quaternary structure - Defined as the three dimensional structure of a multimeric protein composed of several subunits. (i.e the interaction of several proteins) 6) Distinguish between an α helix and a β pleated sheet. • The main chain of a protein is highly polar and therefore hydrophilic (due to the C=O and NH groups. Therefore it seems as though the protein could not fold into an interior protein. However neutralisation of the polar groups is achieved by their H-bonding to form 1 of 2 regular structures: α–helix, β-pleated sheet. • The side chains of individual amino acids project out from within the α–helix. H-bonds between the C=O of one residue and the NH of another residue 4 amino acids along the helix, stabilise the entire structure. There is a occasionally a slight kink in the helical structure, this is due to the presence of proline as one of the amino acids. In proline the last atom of the 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 in the helix, thereby distorting the helical conformation, putting a kink into it. • In the β-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. As with the alpha helix, hydrogen bonds between the NH and C=O groups of two or more b-strands hold the β-pleated sheet together. β-strands can run in the same direction to give a parallel β-pleated sheet, or in opposite directions to give an antiparallel β-pleated sheet. 7) Give examples of the types of bond involved in holding proteins together. • Covalent bonds are the strongest bonds in the protein existing 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 2 atoms bearing negative charges, partially share a positively charged hydrogen. These can occur between atoms on different side chains and the back bone of the protein or between H20 molecules.

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• Ionic interactions, these arise from the electrostatic attraction between charged side chains e.g. Glu, Asp, Lys and Arg. These are relatively strong bonds, particularly when the ion pairs are within the protein interior, excluded from water. (n.b. with a protein most of the charged groups are at the surface of the folded protein, where they can be neutralised by counterions such as salts) • Van der Waals forces are transient weak forces between 2 atoms due to the fluctuating electron cloudsurrounding each atom, leading to temporary electric dipoles. Despite being quite weak individually, cumulatively Van der Waals forces can still have a large part in the conformation of a protein. • Hydrophobic interactions are a major force in the folding of proteins, they force hydrophobic side chains into the interior of the protein, causing a hydrophobic core and a hydrophilic surface to the protein.

Lecture 2: Energetics and enzymes 1) Define the 1st and 2nd Laws of thermodynamics. • 1st Law - Energy can neither be created nor destroyed i.e. it is simply converted from one form to another. • 2nd Law - 2nd Law - In any isolated system, e.g. a single cell or the universe, the degree of disorder can only increase. 2) Describe how oxidation and reduction involve the transfer of electrons. • OIL RIG; oxidation is loss of electrons from a molecule; reduction is gain of electrons to a molecule. • Since the cellular environment is generally aqueous, often, when a molecule gains an electron, it also simultaneously gains a proton eg. A + e- + H+ → AH 3) Explain the concept of free energy and how we can use changes in free energy to predict the outcome of a reaction. • (Gibb’s) Free Energy is defined as the amount of energy within a molecule that could perform useful work at a constant temperature. (units= Kj/mol; symbol= G) • The free energy function combines both the 1st and 2nd Laws of thermodynamics. • Changes in G (ΔG) measure the amount of disorder that results from a particular reaction. A reaction can only occur spontaneously if ΔG is negative. Conversely, a reaction cannot occur spontaneously if ΔG for the reaction is positive. • In the reaction A+B → C+D; ΔG= free energy (C+D) – free energy (A+B) 4) 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. • Pathways within the cell that synthesise molecules are generally energetically unfavourable (e.g. protein synthesis, in order to allow them to take place, the energetically unfavourable reaction is coupled with a highly energically favourable one, providing the overall sum of ΔG as negative- allowing the reaction to proceed Anhydride bonds (in red) link the terminal phosphate groups • Phosphoanhydride bonds have a large –ve ΔG of hydrolysis, and this are said to be “high energy” bonds. The majority of energetically unfavourable reactions are coupled with the energetically favourable hydrolysis of ATP i.e. • ATP → ADP + Pi (ΔG= -7.3 Kcal/mol) • Providing that the sum of the ΔG for the overall reaction is still negative, the reaction will proceed.

5) Describe how enzyemes act as catalysts of reactions with reference to the reaction catalysed by lysosomes • Lysozyme is the component of tears and nasal secretion, it is used in the defence against bacteria, it catalyses the hydrolysis of sugar molecules within bacteria cell walls – which is necessary for the bacteria structure, therefore causing the bacteria to lyse and die. • Lysozyme hydrolyses alternating polysaccharide copolymers of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) which represents the unit of many bacterial cell walls • Lysozyme cleaves at the β(1,4) glycosidic linkage connecting the C1 carbon of NAM to the C4 carbon of NAG • Two acidic residues Glu35 and Asp52 are essential for catalysis.

Non-polar environmen t

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Polar environmen t


• • •

Firstly Glu35 protonates the oxygen in the glycosidic bond, breaking the bond holding the two sugar molecules together. A water enters and is de-protonated by Glu35 (Asp52 stabilises the positive charge in the transition state The hydroxide ion left over, then attacks the remaining NAM sugar molecule adding OH to it. Now both Glu35 and Asp52 are in their original states to continue catalysis. 6) Describe how enzymes act as catalysts of reaction with reference to the reaction catalysed by glucose-6-phosphatase and explain what clinical symptoms are linked to inherited deficiencies of this enzyme. • An enzyme is A protein that acts as a catalyst to induce chemical changes in other substances, itself remaining apparently unchanged by the process • In a biological setting, even those reactions which are energetically favourable do not occur at rates useful for life unless catalysed by enzymes. • • • • • •

Glucose-6-phosphatase catalyses the conversion of glucose-6-phosphate to 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 E nzymes work by bending their substrates in the active site in such a way that the bonds to be broken are stressed and the substrate molecule resembles the transition state (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 ). This makes them more amenable to reaction with other molecules. Von Gierke’s disease sufferers have two mutant G6Pase genes and suffer from hepatomegaly (swollen liver), fasting hypoglycaemia, slow growth and short stature.

7) Outline the differences between lock and key and induced fit models of substrate-enzyme interactions. • Lock and key – in this model the shape of the substrate matches that of the active site of the enzyme. (this explains the specificity of most enzymes for a single substrate. • Induced fit - 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. • The correct model is induced fit, because proteins generally possess a degree of flexibility necessary for function. (proven by crystallography) 8) Describe graphically, the effects of a) substrate concentration, b) temperature and c) pH on enzyme catalysed reactions.

Substrate concentration – the reaction rate tends towards Vmax; at first an increase in substrate concentration has a vast effect on the reaction rate, but the effect decreases as the enzyme becomes increasingly saturated. • Temperature – the rate slowly increases as temperature does until the optimum temperature is reached – the temperature at which the enzyme performs optimally, which varies according to the function and environment that the enzyme is designed for; after this, the enzyme becomes denatured and does not function. • pH – rate is symmetrical about the optimum pH, which once again is dependant on the environment for which the enzyme is designed; above and below this the acidic and basic effects compromise the functioning of the active site and the stability of the protein. 9) 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. • NAD+ (nicotinamide adenine dinucleotide) is a vital component of many dehydrogenation reactions within the body. It has no function on its own but functions only after binding to a protein. NAD+ catalyses the dehydrogenation of substrates by readily accept a hydrogen atom and two electrons. • The substrate glyceraldehyde-3-phosphate reaction is oxidised (hydrogen removed) and also phosphorylated in a coupled reaction.

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• •

Pyruvate is converted into lactate during anaerobic respiration generating free NAD+ used by the muscles 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 dehyrdrogenase oxidises malate to give oxaloacetate, a key metabolite of the TCA cycle.

Lecture 3: Metabolic pathways and ATP production I 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.

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. • 1 molecule of glucose = the use of 2 ATP, the production of 4 ATP (net gain of 2), production of 2 NADH and 2 H+. • Glucose is converted to two triose sugars (which can be inter-converted), one of which can be further phosphorylated to form pyruvate which can enter the Kreb’s (Citric Acid of TCA) cycle.

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3) Define the term substrate level phosphorylation. • 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. 4) Distinguish between aerobic and anaerobic glycolysis with reference to the enzymes involved and the comparative efficiencies of each pathway. • In anaerobic conditions (eg. rapidly contracting muscle) respiration proceeds via the lactate pathway; in aerobic conditions it proceeds via the Kreb’s cycle and oxidative phosphorylation (electron transport chain). • Anaerobic pathway – reducing pyruvate to lactic acid, NAD+ can be regenerated from NADH (oxidation) allowing glycolysis to continue and produce ATP; as lactate is produced it is removed from the cell and moved to the liver where it is converted back to pyruvate – therefore 2 ATP are produced via anaerobic glycolysis. lactate dehydrogenase pyruvate 2NADH + 2H+ •

lactate 2NAD+

Aerobic pathway – involves many more kinase enzymes (catalyse the transfer of a phosphate group from a donor to a substrate) eg. phosphofructokinase and isomerase enzymes eg. triose phosphate isomerase. Aerobic glycolysis means that pyruvate can enter the Kreb’s cycle resulting in the production of up to 38 ATP.

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5) Describe the reactions catalysed by lactate dehydrogenase and creatine kinase and explain the diagnostic relevance of their appearance in plasma. • Lactate dehydrogenase catalyses the inter-conversion of pyruvate and lactate during anaerobic respiration i.e. during intense muscle activity when O2 is a limiting factor. Present in most tissues especially the heart, liver, kidney, skeletal muscle, brain blood cells and lungs. Elevated levels are used to diagnose stroke, heart attack, liver disease (eg hepatitis), muscle injury, muscular dystrophy, and pulmonary infarction. • Creatine kinase is an enzyme that catalyses the inter-conversion of creatine phosphate and ADP to creatine and ATP. Creatine phosphate buffers demands for phosphate. Muscular damage results in leakage of creatine kinase into the bloodstream. Either total levels or the levels of the various isoforms can be detected to diagnose MI, the extent of muscular disease, cause of chest pain and the extent of Duchenne muscular dystrophy. 6) Outline the oxidative phosphorylation reaction catalysed by pyruvate dehydrogenase, with reference to the product and the five co-enzymes required by this enzyme complex. Pyruvate + CoA + NAD+ → acetyl CoA + CO2 + NADH • This is the committed step for the entry of pyruvate into the TCA cycle which occurs in the mitochondrial matrix. The pyruvate dehydrogenase complex is massive, consisting of three enzymes and five co-enzymes: thiamine pyrophosphate (TPP), lipoamide, FAD, CoA, and NAD+. Enzyme Pyruvate Decarboxylase Lipoamide Reductase Transacetylase Dihydrolipoyl Dehydrogenase

(E1) (E2) (E3)

Prosthetic group Thiamine pyrophospate (TPP) Lipoamide FAD (Flavine Adenine Dinucleotide)

Prosthetic groups such as FAD are a permanent part of the complex, whereas NAD+ and other co-enzymes bind reversibly to enzymes. 1.Decarboxylation of pyruvate to give hydroxyethyl (PD & TPP). 2.Oxidation & transfer to lipoamide to give acetylipoamide (LRT). 3.Transfer of the acetyl group to CoA to give acetyl CoA (LRT & CoA). 4.Regeneration of oxidised lipoamide (DD, FAD, NAD+) •

7) Describe the processes by which palmitic acid (fatty acid) and alanine (amino acid) are respectively converted into acetyl Co-A. • Fatty acid metabolism: The oxidation of fatty acids constitutes the most compact fuel for the bodys energy requirements, as a result, fatty acid oxidation yields several times the usual chemical energy that carbohydrates can deliver. (note more than half the body’s energy requirements (including the liver but NOT the brain) comes from fatty acid oxidation, this is enhanced during fasting over long periods. Fatty acids are metabolised in the mitochondria in several stages, firstly they are converted to an acylcoA species AcylCoA synthase

• • • • •

Fatty acid + ATP + HS-CoA AcylCoA + AMP + PPi The acyl species then undergoes β oxidation resulting in the production of acteylCoA and a AcylCoA species (2 carbons shoeter than the original. The β oxidation reactions continue to consequtively remove 2 carbon units from the acylCoA thereby producing acetylCoA (by attaching the carbon units to HS-CoA). On the final cycle 2 acetylCoA’s are produced. Therefore from just 7 β oxidation reactions, the 16 carbon palmitoyl CoA molecule produces 8 acetylCoA molecules. 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 can be 'separated' into pathways depending on the number of carbon atoms the amino acid possesses C3 family e.g. alanine, serine (glycine), and cysteine are all degraded to pyruvate Alanine (C3) is transaminated with α-ketoglutarate meaning that the amine group is transferred from alanine to α-ketoglutarate to produce glutamate and pyruvate. Pyruvate can enter the TCA cycle, while glutamate is re-converted to a-ketoglutarate by glutamate dehydrogenase, generating NH4+ which is ultimately converted to urea alanine aminotransferase Alanine + α-ketoglutarate pyruvate + glutamate (Persistently elevated levels of alanine aminotransferase are a diagnostic for hepatic disorders such as Hepatitis C. )

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Lecture 4: Metabolic pathways and ATP production II 1) Outline the Kreb’s 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 FADH 2 and the cellular location of these reactions. Citrate synthase

Aconitase

Isocitrate dehydrogenase

fumerase H20

α ketogluterate dehydrogenase complex

Succinyl CoA synthetase

the kreb cycle unlike the process of glycolysis takes place in the matrix of the mitochondria, all of the TCA (tricarboxylic acid cycle) enzymes are soluble proteins located in the matrix of mitochondria, except for succinate dehydrogenase, which is found on the inner surface of the mitochondrial inner membrane 2) Calculate the total yield of ATP obtained from the oxidation of one acetyl-CoA molecule by the TCA cycle. • One molecule of acetyl-CoA (x2 for glucose) produces 3 NADH, 1 FADH and a GTP. • Reoxidation of of the reduced co-factors NADH and FADH2 by the process of oxidative phosphorylation yields the following: 3 ATPs per NADH; 2 ATPs per FADH2 and a single ATP for each GTP. • From this we can calculate that 12 ATPs are generated from one revolution of the kreb cycle alone (3NADH+1FADH+1GTP) 3) Compare the oxidation of glucose by the TCA cycle with the beta oxidation of palmitic acid with reference to the ATP produced per molecule of substrate. • Fatty acids are metabolised in the mitochondria in several stages. First they are converted into an acyl-CoA species using up 2 high-energy phosphate bonds (ATP → AMP). Fatty acids are then degraded in a sequence of β-oxidation reactions in which two carbon units are ultimately removed from the carboxyl end of the fatty acid. Enzymes remove two carbons at a time from palmitic acid (16C) and attach them to CoA-SH to form acetyl-CoA which then enters the Kreb’s cycle; palmitic acid can produce 8 molecules of acetyl-CoA, 7 NADH and 7FADH2. (look at equation in lecture 3) • Adding all this together (8x12 + 7x3 + 7x2) fatty acid metabolism yields 131 ATPs, however since 2 high energy phosphate bonds were used to make the initial acylCoA, overall 129 ATPs are yielded from each palmitic acid. • Note that metabolism of palmitic acid yields 5 times ATP than that of oxidation of glucose by the TCA cycle. 4) Outline the glycerol phosphate shunt and the malate-aspartate shunt, in particular stating why these mechanisms are required. The inner mitochondrial membrane is impermeable to NADH and so the use of the glycerol-phosphate shunt and malate-aspartate shunt is needed so that the electrons can be used in oxidative phosphorylation. The glycerol-phosphate shunt:

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Glyceraldehyde-3-phosphate catalyses the cytosolic reaction, whilst the reaction occurring in the mitochondria is catalysed by glycerol dehydrogenase. The malate-aspartate shunt: This takes place primarily in the heart and liver and uses two membrane carriers and four enzymes. Hydrogen is transferred from cytoplasmic NADH to oxaloacetate to give malate (catalysed by malate dehydrogenase). Malate can be transported into the mitochondria where it is rapidly re-oxidised by NAD+ to give oxaloacetate and NADH. The net reaction in terms of NADH is: NADHcytoplasmic + NAD+mitochondrial → NAD+cytoplasmic + NADHmitochondrial 5) Understand the concept of transamination with reference to the malate-aspartate shunt. Transamination - 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. The oxaloacetate formed cannot cross the mitochondrial matrix membrane and so is transaminated by the transfer of an amino group from glutamate to give aspartate which can then cross the membrane via an amino acid carrier and is then reconverted to oxaloacetate through the reverse transamination reaction.

6) Explain in general terms the relationship between TCA intermediates and those pathways involved in amino acid 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, α-ketoglutarate, succinyl CoA, fumarate and oxaloacetate. The amino acids, nucleotides, lipids, sugars, and other molecules become the precursors for the many of the macromolecules of the cell. 7) Give two examples of the use of NADPH in reductive biosynthesis. Like NAD+, NADP+ can pick up two high-energy electrons and in the process, a proton (H+) collectively known as a hydride ion (H-). NADP+ binds to different enzymes than NAD+ because of its slightly different conformation due to the phosphate group attachment. NADPH takes part in anabolic reactions, whereas NADH takes place in catabolic reactions. NADPH is a co-factor in the biosynthesis of RNA; NADPH helps to catalyse the final reaction of several that leads to cholesterol synthesis.

Lecture 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 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. • Evidence supporting the evolutionary origins include i. Mitochondria can only arise from pre-existing mitochondria and chloroplasts. ii. Mitochondria possess their own genome and it resembles that of prokaryotes, being a single circular molecule of DNA, with no associated histones.

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iii.

• • • 2)

Mitochondria have their own protein-synthesizing machinery, which again resembles that of prokaryotes not that of eukaryotes. iv. The first amino acid of their transcripts is always fMet as it is in bacteria (not methionine (Met) that is the first amino acid in eukaryotic proteins). v. 6. 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. Mitochondria are the powerhouse of the cell, generating the bulk of the cellular ATP. They were first shown to contain the enzymes of the Krebs cycle Outer membrane limits the size of the organelle Inner membrane (folds that project inward called cristae) Draw a cross sectional representation of a mitochondrion, and label its component parts.

The reactions of oxidative phosphorylation take place in the inner membrane, in contrast to the Krebs Cycle reactions which occur in the matrix. • Numerous folds within the cristae increase the surface area upon which oxidative phosphorylation can take place. • In some cells e.g. myocytes and sperm, mitochondria distribute themselves where ATP is rapidly consumed. 3) Describe the electron transport chain in mitochondria with reference to the functions of coenzyme Q (ubiquinone) and cytochrome c. • Within the mitochondria, the co-enzymes NADH and FADH2 are re-oxidised by molecular oxygen in the reactions: • NADH + H+ + ½ O2 NAD+ + H20 • FADH2 + ½ O2 FAD + H20 • Each reaction has a ΔG of -52.6 and -40 kcal/mol respectively. (You may recall that ΔG ATP hydrolysis was -7.3 kcal/mol) Thus, the energy released from the re-oxidation of NADH and FADH2 is enough to generate several phosphoanhydride bonds. • Part of this energy is recovered by the components of the electron transport chain and used to synthesise ATP. • Oxidative Phosphorylation proceeds in two steps: i. The translocation or movement of protons from within the matrix of the mitochondria. This is controlled by the electron transport or respiratory chain. ii. The pumped protons are allowed back into the mitochondria through a specific channel, which is coupled to an enzyme which can synthesise ATP (ATP synthase). • The ETC takes places on the cristae and consists of a chain of three complexes and two mobile carriers which act as electron carriers. NADH dehydrogenase complex Cytochrome b-c1 complex Cytochrome oxidase complex Ubiquinone (Co-enzyme Q) Cytochrome C

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Membrane complexes

Mobile carriers


• • • • • •

4) •

These proteins accept electrons and in doing so, a protein (H+) from the aqueous solution. As electrons pass through each of the complexes, a proton is passed or ‘pumped’ to the intermembrane space. (see picture on slides) Ubiquinone can pick up either one or two electrons (together with a 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) In the final electron transfer step, Cytochrome Oxidase receives 4 electrons from cytochrome c and passes them to oxygen to generate water. 4e- + 4H+ + O2 2H2O In addition, 4 protons are also pumped to the intermembrane space, enhancing the proton gradient. Despite being an ideal terminal electron acceptor, reduction of oxygen also leads to the generation of dangerous radicals e.g. superoxide (O2-), the electron transport system has evolved to neutralise this potential problem by having the O2 molecule bound tightly by the active site until all four electrons have been added (O2- is particularly reactive and can cause havoc in biological systems). Outline the chemiosmotic theory. Remember that Oxidative phosphorylation proceeds in two steps: the translocation or movement of protons from within the matrix of the mitochondria. This is controlled by the electron transport or respiratory chain. the pumped protons are allowed back into the mitochondria through a specific channel, which is coupled to an enzyme which can synthesise ATP (ATP synthase). Proton translocation creates an electrochemical gradient across the membrane (both electrical and pH components), energy stored in this proton gradient is used to make ATP by ATP synthase (this is known as the chemiosmotic theory)

ATP synthase consists of two subunits: F0, a proton channel and F1 the enzyme, ATP synthase. Protons re-enter the mitochondrial matrix normally only through the F0 proton channel. The movement of these protons activates ATP synthesis by the

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• • •

F1 subunit. A flow of approximately 3 protons through the ATP synthase are required to make each ATP. Therefore, electron transport and phosphorylation are coupled by the proton gradient. Succinate dehydrogenase is an integral membrane protein that is firmly attached to the inner surface of the inner mitochondrial membrane (from the Kreb cycle).Here, it can communicate directly with ubiquinone. Ubiquinone is the Entry point for Electrons Donated by FADH2 As such one less proton is pumped to the intermembrane space, c.f. NADH and as a consequence, less ATP is produced:

X

FADH2 FAD

5) Explain why carbon monoxide, cyanide, malonate and oliogomycin are poisonous in terms of their effects on specific components of the electron transport chain. • The average human body synthesises around 70kg of ATP per day. • Each of these ATP molecules has a lifespan of between 1 and 5 minutes. • Consequently, any interruption to the process of oxidative phosphorylation and therefore to ATP synthesis, means that a cell rapidly becomes depleted of ATP and is likely to die. • The most common cause of a failure of oxidative phosphorylation is simply a lack of oxygen e.g. hypoxia (diminished), anoxia (total). • Depending on the cell type and their metabolic requirements, death will be within a few minutes (neurons) or a few hours (muscle). • Cyanide (CN-): binds with high affinity to the ferric (Fe3+) form of the haem group in the cytochrome oxidase complex, blocking the flow of electrons through the respiratory chain and thus ATP production. • Carbon monoxide also inhibits electron transfer in cytochrome oxidase, therefore blocking ATP production in a similar fashion to the way cyanide does. • Malonate: closely resembles succinate and acts as a competitive inhibitor of succinate dehydrogenase. This is the one Kreb’s cycle enzyme that resides in the inner mitochondrial membrane and passes its electrons directly to ubiquinone via FAD. Malonate effectively slows down the flow of electrons from succinate to ubiquinone by inhibiting the oxidation of succinate to fumerate. • Oligomycin is an antibiotic (produced by Streptomyces) that inhibits oxidative phosphorylation by bind with the ‘stalk’ of ATP synthase and in doing so, blocks the flow of protons through the enzyme. As a result, ATP synthesis in inhibited and a backlog of protons will build up in the intermembrane space. This, in turn, will eventually inhibit the flow of electrons through the ETC as the [H+] outside the mitochondrion will build up to saturation point at which no more protons can be pumped out against this proton gradient. 6) Describe how oxidative phosphorylation can be measured experimentally. • The oxygen electrode measures the concentration of oxygen in a solution contained in the chamber of the apparatus. A suspension of mitochondria from homogenized tissue is incubated within a sealed incubation chamber containing an isotonic medium containing substrate eg. succinate and Pi. The addition of ADP causes a sudden burst of oxygen uptake as the ADP is converted to ATP. This is termed coupled respiration. By adding various substances to the chamber we can determine their effects on oxidative phosphorylation.

Lecture 6: Lipids and membranes 1) Describe the structure of: fatty acids, triglycerides, phospholipids, cholesterol, and sphingomyelin.

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Fatty acids that occur in natural fats are straight-chain derivatives and contain an even number of carbon atoms because they are synthesised from two-carbon units. The chain may be saturated (containing no double bonds) or unsaturated (containing one or more double bonds). These are the simplest lipids and are usually constituents of more complex lipids.


headgroup containing phosphate (hydrophilic)

o o c=o c=o

glycerol backbone

Phospholipids have a headgroup containing phosphate which is hydrophilic, and backbone of glycerol and two fatty acid tails which are hydrophobic. The phosphate is esterified with the –OH of a suitable alcohol.

Triacylglycerols (triglycerides) are esters of the alcohol glycerol and fatty acids. In naturally occurring fats, the proportion of triacylglycerol molecules containing the same fatty acid residue in all three ester positions is very small. They are nearly all mixed acylglycerols

tails - consisting of two fatty acid chains (hydrophobic)

• Cholesterol is a derivative of saturated tetracyclic hydrocarbon. All cyclohexane rings are in the chain structure which means they have a plain, rigid structure. The storage form is acylated at position 3.

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Sphingomyelins are found in large quantities in brain and nervous tissue. OH hydrolysis the sphingomyelins yield a fatty acid, phosphoric acid, choline and a complex amino alcohol, sphingosine. The combination of sphingosine plus fatty acid is known as ceramide.


2) Give examples of how the lipid composition can differ for different cellular membranes, and indicate the significance of this. • The degree of fluidity of a cell membrane is important for membrane function and has to be maintained within certain limits. This depends on its phospholipid composition and, especially, on the nature of the hydrocarbon tails. • The closer and more regular the packing of the hydrocarbon tails, the more viscous and less fluid the bilayer will be. Length and unsaturation are two properties of phospholipid hydrocarbon tails that affect how packed the tails will be. • Cholesterol molecules fill the spaces between neighbouring phospholipid molecules caused by kinks in their unsaturated hydrocarbon tails. • Cholesterol thus stiffens the bilayer making it less fluid and less permeable. • Different membranes within the cell and between cells have different compositions, as reflected in the ratio of protein to lipid. Specific deficiencies or alterations of certain membrane components lead to a variety of diseases. • Normal cellular function depends on normal membranes. 3) Outline the pathway for synthesis of fatty acids. Acetyl CoA is the key intermediate between fat and carbohydrate metabolism. • Step 1 – Production of Malonyl CoA catalysed by Acetyl CoA carboxylase. • Step 2 – Activation by acyl carrier protein. Similar to CoA activation in β–oxidation. • Step 3 – Elongation by successive addition of two-carbon units catalysed by FA synthase. • There is a striking similarity between fatty acid degradation and synthesis. • Overall reaction: Acetyl CoA (C2) + 7 Malonyl CoA C3 + 14 NADPH + 14 H+ ↔ Palmitate C16 + 7 HCO2 + 6 H2O + 8 CoA SH + 14 NADP+ 4) Distinguish between the pathways for synthesis and metabolism of fatty acids in terms of: substrates and products, coenzymes used, carrier molecules, cellular location. Fatty acids are metabolised in the mitochondria in several stages in the cytosol (lipogenesis). 1. First they are converted into an acyl-CoA species involving 2 high-energy phosphate bonds (ATP → AMP + PPi) and the enzyme acyl CoA synthase. 2. Fatty acids are then degraded in a sequence of β-oxidation reactions in which two carbon units are ultimately removed from the carboxyl end of the fatty acid involving FAD and NAD+ being reduced to FADH2 and NADH. 3. Enzymes remove two carbons at a time from palmitic acid (16C) and attach them to CoA-SH to form acetyl-CoA which then enters the Kreb’s cycle. Note that in fatty acid synthesis NADP electron carrier is used whereas in the degradation, NAD+ and FAD is used. Also lipogenesis is carried out in the cytosol as opposed to the mitochondria, which is where lipid degradation is carried out.

Lecture 7: Cholesterol 1) Explain the physiological functions of cholesterol in membrane stability. • High cholesterol leads to reduced fluidity due to reducing phase transitions of lipids and reducing lateral motility of polar lipids. Cholesterol thus stiffens the bilayer making it less fluid and less permeable.

2) Outline the synthesis of cholesterol from acetate. Five stages: • Mevalonate, a six-carbon compound, is synthesized from acetyl-CoA. • Isoprenoid units are formed from mevalonate by loss of CO2. • Six isoprenoid units condense to form the intermediate, squalene. • Squalene cyclizes to give rise to the parent steroid, lanesterol. • Cholesterol is formed from lanesterol after several further steps, including the loss of 3 methyl groups. 3) Outline the synthesis of bile acids and steroid hormones from cholesterol. • Bile acids - The 7α-hydroxylation of cholesterol is the first committed step in the biosynthesis of bile acids, and it is this reaction that is rate-limiting in the pathway for synthesis of the acids. The reaction is catalysed by 7α-hydroxylase and requires oxygen,

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NADPH, and cytochrome P450 which is a monooxygenase. Subsequent hydroxylation steps are also catalysed by monooxygenases. These hydroxylation reactions and shortening of the side chain gives rise to the typical bile acid structures of α–OH groups on positions 3 and 7 and full saturation of the steroid nucleus. Conjugated bile acids- taurocholate and glycocholate. Bile salts go on to emulsify dietary fats • Steroid hormones – All mammalian steroid hormones are formed from cholesterol via pregnenolone (produced from cholesterol by cholesterol desmolase) through a series of reactions that occur in either the mitochondria or endoplasmic reticulum of the adrenal cell. Hydroxylases that require molecular oxygen and NADPH are essential, and dehydrogenases, an isomerase, and a lyase reaction are also necessary for certain steps. 4) Describe the mechanism of transport of cholesterol around the body and its uptake into cells. • Cholesterol is extremely insoluble and is transported in the bloodstream bound to protein in the form of particles called lowdensity lipoproteins, or LDL. • The LDL binds to receptors located on cell surfaces, and the receptor-LDL complexes are ingested by receptor-mediated endocytosis and delivered to endosomes. • The interior of endosomes is more acid than the surrounding cytosol or the extracellular fluid, and in this acidic environment the LDL dissociates from its receptor. • The receptors are returned in transport vesicles to the plasma membranes for reuse, while the LDL is delivered to lysosomes. • In the lysosomes hydrolytic enzymes break down the LDL. • The cholesterol is released and escapes into the cytosol, where it is available for new membrane synthesis. • The LDL receptors on the cell surface are continually internalized and recycled, where they are occupied by LDL or not. 5) Draw a diagram of low density lipoprotein (LDL).

6) Explain why disturbances in cholesterol homeostasis cause disease. • Hypercholesterolaemia causes disease. Familial hypercholesterolaemia occurs in people with a genetic defect causing a lack of LDL receptors, which remove cholesterol from the bloodstream. • Cholesterol is a constituent of gall stones. • Numerous studies have shown a correlation between coronary heart disease, blood cholesterol and the consumption of fat. • Cholesterol is found only in food of animal origin and particularly in egg yolk. • Atherosclerosis is characterised by the deposition of cholesterol and cholesteryl ester of lipoproteins containing apo B-100 in the connective tissue of the arterial walls. 7) Give an example of how a selective enzyme inhibitor can be used as a pharmacological agent controlling cholesterol metabolism. • HMGCoA reductase inhibitors (eg. mevastatin and lovastatin) reduce LDL cholesterol levels by up-regulation of the LDL receptors.

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FH= Familial Hypercholesterolaemia

Lecture 8: Membrane Trafficking 1) Explain the terms “endocytosis” and “exocytosis”. • Endocytosis – In endocytosis, the cell engulfs a drop of the extracellular fluid. It does this by folding inward a portion of its plasma membrane. The pouch that results is pinched off from the plasma membrane forming a membrane-enclosed bubble-like vesicle. • Exocytosis – a process whereby newly made proteins, lipids and carbohydrates are delivered from the ER, via the Golgi apparatus, to the cell surface by transport vesicles that fuse with the plasma membrane.

2) Describe the pathway and cellular locations for synthesis, post-translational modification and exocytosis of a secreted protein. • The ER is arranged as sheets or plates, as a tubular network or a combination of both. • RER have ribosomes on cytoplasmic surface, which synthesise proteins into the lumen. • SER have no ribosomes and secret lipid soluble components e.g. steroid hormones, from the lumen. From the lumen the molecules travel in vesicles to the cis face of the Golgi apparatus. • The Golgi is a relatively ordered stack of smooth cisternae and processes the membrane and its contents in relation to destination and function. • From the trans face of the Golgi apparatus, secretary vesicles bud off and then fuse with the cytoplasm releasing the vesicle contents to the extracellular environment.

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3) Distinguish “constitutive” and “regulated” secretion. • Exocytosis is either regulated secretion (vesicles stored until a stimulatory signal triggers exocytosis) or constitutive secretion (immediate/continuous exocytosis). 4) Describe the process of receptor-mediated endocytosis and the roles played by endocytic vesicles, early endosomes, late endosomes, and lysosomes. • Macromolecules bind to complementary receptors on the cell surface and enter the cell as receptor-macromolecule complexes in clathrin-coated vesicles. This provides a selective concentrating mechanism that increases the efficiency of internalisation of particular macromolecules more than 1000-fold compared with ordinary pinocytosis.

An example is the uptake of cholesterol: • Cholesterol is extremely insoluble and is transported in the bloodstream bound to protein in the form of particles called lowdensity lipoproteins, or LDL. • The LDL binds to receptors located on cell surfaces, and the receptor-LDL complexes are ingested by receptor-mediated endocytosis and delivered to endosomes. • The interior of endosomes is more acid than the surrounding cytosol or the extracellular fluid, and in this acidic environment the LDL dissociates from its receptor. • The receptors are returned in transport vesicles to the plasma membranes for reuse, while the LDL is delivered to lysosomes. In the lysosomes hydrolytic enzymes break down the LDL. • The cholesterol is released and escapes into the cytosol, where it is available for new membrane synthesis. • The LDL receptors on the cell surface are continually internalised and recycled, where they are occupied by LDL or not. 5) Give a general description of the molecular mechanisms of vesicular transport within cells. 1. Cargo sorting and vesicle formation at the donor membrane. 2. Vesicle movement propelled by motor inside cells (microtubules and actin filaments). 3. Vesicle tethering/docking with specific receptors on acceptor membrane. 4. Vesicle fusion with acceptor membrane 6) Give examples of diseases resulting from defects in the secretary and endocytic pathways. • Familial hypercholesterolaemia – genetic mutation resulting in a defect in the LDL receptor of the cell. • Intracellular trafficking is involved in many diseases including: - Genetic (> 75 genetic diseases or syndromes) - Infection - cancer

Lecture 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.

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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. 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.

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.

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Lactate/alanine pass into the blood and the liver uses them to replenish glucose by gluconeogenesis

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Jig's Metabolism