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Aim: predict the direction of a reaction. 1. Delta G = Delta H - T Delta S. A negative Delta G means the reaction can proceed spontaneously (exergonic). A positive value means it needs energy (endergonic). 2. Delta G depends on the actual concentrations: Delta G = Delta G0' + RT ln([products]/[reactants]). Delta G0' is the standard value at pH 7 and 1 M. 3. At equilibrium Delta G = 0. 4. Free-energy changes of sequential reactions add up. An endergonic step can be driven by coupling it to a strongly exergonic one that shares an intermediate. Exam trap: an enzyme lowers the activation energy but never changes Delta G or the equilibrium position. Clinical tip: many pathway steps are near equilibrium in the cell (Delta G close to 0), while regulated steps are far from equilibrium and irreversible. That is why regulation acts on them.

Structure: adenine, ribose and three phosphates joined by two phosphoanhydride bonds. Why the hydrolysis releases energy - Electrostatic repulsion between the negative phosphates is relieved. - The products (ADP and phosphate) are stabilised by resonance and hydration. - Standard Delta G0' of ATP -> ADP + Pi is about -30.5 kJ/mol; in the cell it is about -50 kJ/mol because ATP is kept far above equilibrium. ATP works by group transfer: it phosphorylates a substrate, which raises the substrate's free energy and drives the next step. Other high-energy compounds: phosphoenolpyruvate, 1,3-bisphosphoglycerate and phosphocreatine have a more negative hydrolysis energy than ATP, so they can phosphorylate ADP. Glucose 6-phosphate has a less negative value. Clinical tip: ATP turns over about its own body weight per day. It cannot be stored, so it must be regenerated continuously.

Thioesters: the bond between an acyl group and the sulfur of coenzyme A releases a large amount of free energy on hydrolysis (about -31 kJ/mol), similar to ATP. Acetyl-CoA is therefore an activated acetyl group. Fuel oxidation: the more reduced a carbon is, the more energy its oxidation releases. Fats (highly reduced) give about 9 kcal/g and carbohydrates and proteins about 4 kcal/g. Electron flow: electrons removed from fuels are carried by NADH and FADH2 to the respiratory chain, where the flow to oxygen drives ATP synthesis. Exam trap: ATP is not made directly by fuel oxidation. Oxidation reduces coenzymes, and the energy is converted to ATP later.

Electron carriers - NAD+ (from niacin, vitamin B3) accepts a hydride ion to become NADH. NAD+/NADH is used mainly in catabolism (dehydrogenases). - NADP+/NADPH has an extra phosphate. NADPH is the reductant of biosynthesis (fatty acids, cholesterol) and of antioxidant defence. - FAD (from riboflavin, B2) and FMN carry two electrons and two protons. They are tightly bound to their enzymes (flavoproteins), for example succinate dehydrogenase. Group carrier - Coenzyme A (from pantothenic acid, B5) carries acyl groups as thioesters. Acetyl-CoA is the hub of fuel metabolism. Other activated carriers: ATP (phosphoryl), biotin (CO2), tetrahydrofolate (one-carbon units), thiamine pyrophosphate and lipoate (acyl in dehydrogenase complexes). Clinical tip: deficiency of niacin causes pellagra (dermatitis, diarrhoea, dementia), and thiamine deficiency causes beriberi and the Wernicke syndrome in alcoholics.

1. Define exergonic and endergonic and say how a cell drives an endergonic reaction. 2. Why does an enzyme not change the equilibrium of a reaction? 3. Give two reasons why ATP hydrolysis is strongly exergonic. 4. Name the coenzyme mainly used in biosynthesis and the one mainly used in catabolism. 5. Which vitamins give NAD, FAD and coenzyme A? 6. What is special about the thioester bond of acetyl-CoA?

1. Exergonic: negative Delta G, spontaneous. Endergonic: positive Delta G. The cell couples the endergonic step to ATP hydrolysis or another exergonic step through a shared intermediate. 2. An enzyme lowers activation energy for both directions equally. Delta G and the equilibrium constant depend only on the reactants and products. 3. Relief of charge repulsion between phosphates, and stabilisation of the products by resonance and hydration. 4. NADPH for biosynthesis, NAD+/NADH for catabolism. 5. Niacin (B3), riboflavin (B2), pantothenic acid (B5). 6. Its hydrolysis releases as much free energy as ATP, so acetyl-CoA is an activated acetyl donor.

Aim: recognise the main sugars and their forms. 1. Empirical formula (CH2O)n. Aldoses carry an aldehyde (glucose, galactose) and ketoses a ketone (fructose). 2. D and L forms are mirror images. Nearly all natural sugars are D. 3. Epimers differ at one carbon: glucose and galactose differ at C4, glucose and mannose at C2. 4. In solution hexoses cyclise into pyranose (six-membered) or furanose (five-membered) rings. The new hydroxyl at the anomeric carbon gives alpha and beta anomers, which interconvert (mutarotation). 5. The anomeric carbon is a reducing group when free. Derivatives: sugar phosphates (glucose 6-phosphate), amino sugars (glucosamine, N-acetylglucosamine), acidic sugars (glucuronic acid), sugar alcohols (sorbitol) and deoxy sugars (2-deoxyribose in DNA). Clinical tip: sorbitol accumulation in the lens contributes to diabetic cataract.

A glycosidic bond forms by condensation between the anomeric carbon of one sugar and a hydroxyl of another. - Maltose: glucose alpha(1-4) glucose. Reducing. - Lactose: galactose beta(1-4) glucose. Reducing. Milk sugar. - Sucrose: glucose alpha(1-2) fructose beta. Non-reducing, because both anomeric carbons are used in the bond. - Trehalose: glucose alpha(1-1) glucose, non-reducing. Exam trap: alpha versus beta matters for digestion. Human enzymes cleave alpha(1-4) bonds of starch but not the beta(1-4) bonds of cellulose. Clinical tip: lactose intolerance results from lactase deficiency. Undigested lactose is fermented by colonic bacteria and causes bloating and diarrhoea.

Storage homopolysaccharides (glucose polymers) - Starch (plants): amylose, unbranched alpha(1-4), and amylopectin, with alpha(1-6) branches about every 24-30 residues. - Glycogen (animals): like amylopectin but branched every 8-12 residues. Many ends allow fast mobilisation. Stored in liver and muscle. Structural homopolysaccharides - Cellulose: beta(1-4) glucose, straight chains that form fibres by hydrogen bonds. Indigestible for humans, so it acts as dietary fibre. - Chitin: beta(1-4) N-acetylglucosamine, in arthropod shells and fungal walls. Heteropolysaccharides: glycosaminoglycans are unbranched chains of repeating disaccharides, usually with sulfate and carboxyl groups (hyaluronic acid, chondroitin sulfate, heparin, keratan sulfate). They bind water, resist compression and form cartilage and synovial fluid. Clinical tip: heparin, a highly sulfated glycosaminoglycan, is an anticoagulant.

- Proteoglycans: a core protein carrying many glycosaminoglycan chains. Found in the extracellular matrix and cartilage. - Glycoproteins: proteins with branched oligosaccharides, attached to Asn (N-linked) or Ser/Thr (O-linked). They function in folding, stability, cell recognition and immune responses. - Glycolipids: lipids with an oligosaccharide head, on the outer leaflet of the plasma membrane. The ABO blood group antigens are oligosaccharides on glycoproteins and glycolipids. Clinical tip: differences in a terminal sugar (N-acetylgalactosamine, galactose, or none) define blood groups A, B and O.

1. What separates an aldose from a ketose? Give one example of each. 2. How do glucose and galactose differ? What is that relationship called? 3. Why is sucrose a non-reducing sugar? 4. Compare amylopectin and glycogen. 5. Why can humans not digest cellulose? 6. What is the difference between a proteoglycan and a glycoprotein?

1. Aldose: aldehyde group (glucose). Ketose: ketone group (fructose). 2. They differ at C4 only. They are epimers. 3. The bond involves both anomeric carbons, so neither has a free reducing group. 4. Both are alpha(1-4) chains with alpha(1-6) branches. Glycogen is more branched (every 8-12 residues) than amylopectin (24-30). 5. Cellulose has beta(1-4) bonds and humans lack a beta-glucosidase for them. 6. A proteoglycan is mostly carbohydrate (long glycosaminoglycan chains on a core protein). A glycoprotein is mostly protein with short branched oligosaccharides.

Fatty acids: a carboxyl group and a hydrocarbon chain, usually 12-24 carbons and even in number. - Saturated: no double bonds (palmitic 16:0, stearic 18:0). Straight chains pack tightly, so they are solid at room temperature. - Unsaturated: cis double bonds put kinks in the chain, lowering the melting point (oleic 18:1, linoleic 18:2, linolenic 18:3). - Omega-3 and omega-6: position of the first double bond from the methyl end. Linoleic (omega-6) and alpha-linolenic (omega-3) are essential because humans cannot insert double bonds beyond carbon 9. Triacylglycerols: glycerol esterified with three fatty acids. They are nonpolar, stored anhydrous in adipocytes and give about 9 kcal/g, more than twice the energy of carbohydrate per gram. Clinical tip: trans fats raise LDL and lower HDL cholesterol and are associated with cardiovascular risk.

Amphipathic lipids have a polar head and nonpolar tails, so in water they form bilayers. - Glycerophospholipids: glycerol, two fatty acids, phosphate and a head group (choline in phosphatidylcholine, serine, ethanolamine, inositol). Phosphatidylinositol is a source of second messengers. - Sphingolipids: built on sphingosine. Sphingomyelin has a phosphocholine head. Glycolipids (cerebrosides, gangliosides) carry sugars instead. - Cholesterol: a rigid steroid ring that inserts between the tails, reducing fluidity at high temperature and preventing packing at low temperature. Clinical tip: dipalmitoyl phosphatidylcholine is the main component of lung surfactant. Its deficiency causes respiratory distress syndrome in premature newborns.

Steroids share a four-ring nucleus. Cholesterol (27 carbons) gives rise to: - steroid hormones (cortisol, aldosterone, oestrogens, progesterone, androgens) - bile acids, which emulsify dietary fat - vitamin D Other roles - Eicosanoids (prostaglandins, thromboxanes, leukotrienes) are derived from arachidonic acid and act locally in inflammation and clotting. Aspirin inhibits cyclooxygenase. - Fat-soluble vitamins A, D, E and K. - Lipoproteins transport lipids in blood. Exam trap: humans cannot break the steroid ring, so cholesterol leaves the body only as bile acids or as free cholesterol in bile.

1. Why do cis double bonds lower the melting point of a fatty acid? 2. Which fatty acids are essential and why? 3. Why are triacylglycerols a better energy store than glycogen? 4. Draw the general organisation of a phospholipid. 5. What does cholesterol do in the membrane? 6. Name three products made from cholesterol.

1. Cis bonds kink the chain, so the molecules pack loosely and need less energy to melt. 2. Linoleic (omega-6) and alpha-linolenic (omega-3), because humans cannot introduce double bonds beyond C9. 3. They are anhydrous and more reduced, giving over twice the energy per gram, while glycogen binds water. 4. A polar head group and phosphate on a glycerol (or sphingosine) backbone with two nonpolar fatty acid tails. 5. It regulates fluidity: it stiffens the membrane at high temperature and prevents tight packing at low temperature. 6. Steroid hormones, bile acids and vitamin D.

Common structure: alpha carbon bound to an amino group, a carboxyl group, hydrogen and a variable R group. Except glycine, the alpha carbon is chiral, and proteins contain only L-amino acids. Classes by side chain 1. Nonpolar aliphatic: glycine, alanine, valine, leucine, isoleucine, methionine, proline (a ring that restricts the backbone). 2. Aromatic: phenylalanine, tyrosine, tryptophan. 3. Polar uncharged: serine, threonine, cysteine (forms disulfide bridges), asparagine, glutamine. 4. Positive (basic): lysine, arginine, histidine (imidazole, pKa near 6, buffers at physiological pH). 5. Negative (acidic): aspartate, glutamate. At neutral pH free amino acids are zwitterions. The isoelectric point (pI) is the pH where the net charge is zero. Clinical tip: phenylketonuria is a lack of phenylalanine hydroxylase, so phenylalanine accumulates and must be restricted in the diet.

A peptide bond forms by condensation between the carboxyl of one amino acid and the amino group of the next, releasing water. A chain has an N-terminus (free amino group) and a C-terminus (free carboxyl), and is read from N to C. Properties of the peptide bond 1. It has partial double-bond character (resonance), so it cannot rotate. 2. The six atoms around it lie in one plane. 3. It is uncharged, but its C=O and N-H groups form hydrogen bonds. 4. It is almost always trans, except before proline, where cis is possible. Only the phi and psi angles around the alpha carbon are free, and only some combinations are allowed (Ramachandran plot). Primary structure is the amino-acid sequence, specified by the gene. A single change can alter function, as in sickle-cell disease (Glu6Val in the beta chain of haemoglobin).

Alpha helix: a right-handed coil with 3.6 residues per turn. Each C=O forms a hydrogen bond with the N-H four residues along. Side chains point outward. Proline breaks helices. Beta sheet: extended strands lying side by side, held by hydrogen bonds between strands. Strands can be parallel or antiparallel. Tertiary and quaternary structure: the three-dimensional fold of a whole chain (hydrophobic collapse, ionic bonds, disulfide bridges) and the association of several chains, as in haemoglobin (four subunits). Structure equals function: loss of native structure (denaturation) by heat, pH or urea destroys function. Not covered in these notes: the later levels of protein structure and folding diseases, which are not taught in the slides.

1. Draw the general formula of an amino acid. 2. Which amino acids are aromatic? 3. What is the isoelectric point? 4. Why is the peptide bond planar? 5. Describe the alpha helix: turns, hydrogen bonds and orientation of side chains. 6. What amino-acid change causes sickle-cell disease?

1. Central alpha carbon with NH3+, COO-, H and R. 2. Phenylalanine, tyrosine, tryptophan. 3. The pH at which the net charge is zero. 4. Resonance gives the C-N bond partial double-bond character. 5. Right-handed, 3.6 residues per turn, C=O bonded to N-H four residues ahead, side chains outward. 6. Glutamate to valine at position 6 of the beta-globin chain.

- Purines: adenine and guanine (two rings). - Pyrimidines: cytosine, thymine (DNA) and uracil (RNA) (one ring). - Sugar: ribose in RNA, 2-deoxyribose in DNA. - Nucleoside = base + sugar. Nucleotide = nucleoside + phosphate at C5. Nucleotides are joined by 3'-5' phosphodiester bonds, so a strand has a 5' end and a 3' end and is written 5' to 3'. Tip for names: adenosine, guanosine, cytidine, uridine and thymidine are nucleosides. AMP, GTP, dCTP are nucleotides.

DNA double helix (Watson and Crick, using Franklin's diffraction data and Chargaff's rules) - Two antiparallel strands, sugar-phosphate backbones outside, bases inside. - A pairs with T (two hydrogen bonds) and G pairs with C (three). G/C-rich DNA is more stable. - B-DNA is the common right-handed form (10.5 bp per turn). A-DNA is a shorter, wider right-handed form. Z-DNA is left-handed. RNA: single stranded, ribose, uracil instead of thymine. Main types: mRNA (message), tRNA (adapter with an anticodon), rRNA (ribosome structure and catalysis), plus small regulatory RNAs. Other functions of nucleotides: ATP and GTP as energy carriers, cyclic AMP and cyclic GMP as second messengers, and components of coenzymes (NAD, FAD, CoA).

1. Name the purines and pyrimidines. 2. What is the difference between a nucleoside and a nucleotide? 3. Why are the DNA strands called antiparallel? 4. How many hydrogen bonds join A-T and G-C? 5. Which RNA carries the anticodon?

1. Purines: A, G. Pyrimidines: C, T, U. 2. A nucleoside is base plus sugar. A nucleotide adds one or more phosphates. 3. One runs 5' to 3' and the other 3' to 5'. 4. Two and three. 5. tRNA.

Location: cytosol. Two phases. Preparatory phase (spends 2 ATP) 1. Hexokinase (glucokinase in liver) phosphorylates glucose to glucose 6-phosphate. 2. Isomerase gives fructose 6-phosphate. 3. Phosphofructokinase-1 (PFK-1) gives fructose 1,6-bisphosphate. This is the main control point. 4. Aldolase splits it into DHAP and glyceraldehyde 3-phosphate, which are interconverted by triose phosphate isomerase. Payoff phase (per triose) 5. Glyceraldehyde 3-phosphate dehydrogenase makes 1,3-bisphosphoglycerate and NADH. 6. Phosphoglycerate kinase makes 3-phosphoglycerate and ATP (substrate-level phosphorylation). 7. Mutase and enolase give phosphoenolpyruvate. 8. Pyruvate kinase gives pyruvate and ATP. Balance: glucose + 2 NAD+ + 2 ADP + 2 Pi -> 2 pyruvate + 2 NADH + 2 ATP + 2 H2O + 2 H+. Exam trap: 4 ATP are made and 2 are spent, so the net gain is 2 ATP. Clinical tip: pyruvate kinase deficiency causes haemolytic anaemia, because red cells depend entirely on glycolysis.

Galactose: galactokinase makes galactose 1-phosphate, which is converted to UDP-galactose and then to UDP-glucose and glucose 1-phosphate. Galactosaemia results from a defect in galactose 1-phosphate uridyltransferase and causes cataract, liver damage and intellectual disability in infants fed milk. Fructose: in liver, fructokinase makes fructose 1-phosphate, which aldolase B splits into DHAP and glyceraldehyde. This bypasses the PFK-1 control step, so fructose is metabolised rapidly. Aldolase B deficiency causes hereditary fructose intolerance.

Site: mainly liver, also kidney cortex. Precursors: lactate, glycerol, glucogenic amino acids (alanine especially) and propionate. Fatty acids cannot make net glucose in humans. The bypass reactions 1. Pyruvate to phosphoenolpyruvate: pyruvate carboxylase (mitochondria, needs biotin) makes oxaloacetate, then PEP carboxykinase. 2. Fructose 1,6-bisphosphate to fructose 6-phosphate: fructose 1,6-bisphosphatase. 3. Glucose 6-phosphate to glucose: glucose 6-phosphatase (liver, in the endoplasmic reticulum). Cost: 4 ATP and 2 GTP plus 2 NADH per glucose. It is expensive but essential during fasting to feed the brain and red cells. Clinical tip: glucose 6-phosphatase deficiency (von Gierke disease) causes severe fasting hypoglycaemia and hepatomegaly.

Glycolysis - Hexokinase is inhibited by glucose 6-phosphate. Liver glucokinase has a high Km and is not inhibited, so the liver acts as a glucose sensor. - PFK-1 is inhibited by ATP and citrate and activated by AMP and fructose 2,6-bisphosphate. - Pyruvate kinase is activated by fructose 1,6-bisphosphate and, in liver, inhibited by phosphorylation (glucagon) and by alanine. Gluconeogenesis - Fructose 2,6-bisphosphate inhibits fructose 1,6-bisphosphatase and activates PFK-1. Insulin raises it and glucagon lowers it. - Acetyl-CoA activates pyruvate carboxylase. Hormones: insulin favours glycolysis, glucagon favours gluconeogenesis in liver. Glucose transporters: GLUT1 (most cells), GLUT2 (liver, pancreatic beta cell, high Km), GLUT3 (neurons), GLUT4 (muscle and fat, insulin dependent), GLUT5 (fructose).

Location: cytosol; active in liver, adipose tissue, adrenal cortex, red cells and dividing cells. Oxidative phase: glucose 6-phosphate dehydrogenase (the regulated, rate-limiting step) converts glucose 6-phosphate to 6-phosphogluconolactone and generates NADPH. Further steps give ribulose 5-phosphate and a second NADPH plus CO2. Non-oxidative phase: reversible transketolase and transaldolase reactions interconvert pentoses and glycolytic intermediates, so the cell can make ribose when needed or recycle it into glycolysis. Clinical tip: G6PD deficiency leaves red cells unable to make enough NADPH to keep glutathione reduced. Oxidant drugs, fava beans and infections cause haemolysis.

1. How many ATP are spent and made in glycolysis? What is the net yield? 2. Which glycolytic step is the main control point and what regulates it? 3. Name the three irreversible steps of glycolysis and the enzymes that bypass them in gluconeogenesis. 4. Why is glucokinase useful in the liver? 5. Which products does the pentose phosphate pathway supply? 6. Which precursors cannot give net glucose?

1. 2 ATP are spent and 4 made, so the net is 2 ATP (plus 2 NADH). 2. PFK-1. ATP and citrate inhibit; AMP and fructose 2,6-bisphosphate activate. 3. Hexokinase (glucose 6-phosphatase), PFK-1 (fructose 1,6-bisphosphatase), pyruvate kinase (pyruvate carboxylase plus PEP carboxykinase). 4. Its high Km lets the liver take up glucose only when blood glucose is high, and it is not inhibited by glucose 6-phosphate. 5. NADPH and ribose 5-phosphate. 6. Fatty acids (even-chain), because acetyl-CoA cannot be converted to pyruvate.

Breakdown (glycogenolysis) 1. Glycogen phosphorylase removes glucose from non-reducing ends as glucose 1-phosphate (uses phosphate, not water). It needs pyridoxal phosphate. 2. The debranching enzyme moves three residues from a branch and cuts the alpha(1-6) bond releasing free glucose. 3. Phosphoglucomutase converts glucose 1-phosphate to glucose 6-phosphate. 4. Liver has glucose 6-phosphatase and releases free glucose. Muscle lacks it and uses the glucose 6-phosphate itself. Synthesis (glycogenesis) 1. UDP-glucose pyrophosphorylase makes UDP-glucose. 2. Glycogenin primes the chain. 3. Glycogen synthase extends alpha(1-4) chains. 4. The branching enzyme makes alpha(1-6) branches. Clinical tip: glycogen storage diseases include von Gierke (glucose 6-phosphatase), Pompe (lysosomal alpha-glucosidase), Cori (debranching enzyme) and McArdle (muscle phosphorylase, exercise cramps).

Breakdown is activated by glucagon (liver) and adrenaline (liver and muscle) through cyclic AMP and protein kinase A, which activates phosphorylase kinase, which converts phosphorylase b to the active a form. In muscle, calcium and AMP also activate phosphorylase. Synthesis is favoured by insulin, which activates protein phosphatase 1. Phosphorylation inactivates glycogen synthase, and dephosphorylation activates it. So the same kinase cascade turns degradation on and synthesis off. Glucose itself, in liver, binds phosphorylase a and promotes its dephosphorylation. Exam trap: liver glycogen serves the whole body, muscle glycogen serves only the muscle.

1. Which enzyme removes glucose from glycogen and what is the product? 2. What does the debranching enzyme do? 3. Why can muscle not release glucose into the blood? 4. Which hormones activate phosphorylase? 5. Which enzyme deficiency causes McArdle disease?

1. Glycogen phosphorylase, giving glucose 1-phosphate. 2. It transfers three residues of a branch to the main chain and hydrolyses the alpha(1-6) bond. 3. Muscle lacks glucose 6-phosphatase. 4. Glucagon (liver) and adrenaline. 5. Muscle glycogen phosphorylase.

Anaerobic: lactate dehydrogenase converts pyruvate to lactate and regenerates NAD+, so glycolysis continues. Red cells and exercising muscle rely on this. Yeast makes ethanol and CO2. Cori cycle: muscle lactate goes to the liver, which converts it to glucose and returns it to muscle. Warburg effect: many tumour cells ferment glucose to lactate even with oxygen available. Aerobic: the pyruvate dehydrogenase complex (in the mitochondrial matrix) converts pyruvate to acetyl-CoA, CO2 and NADH. It needs five cofactors: thiamine pyrophosphate, lipoate, CoA, FAD and NAD+. It is inhibited by acetyl-CoA, NADH and ATP, and by phosphorylation. Clinical tip: thiamine deficiency impairs pyruvate dehydrogenase and causes lactic acidosis.

Location: mitochondrial matrix. 1. Acetyl-CoA + oxaloacetate -> citrate (citrate synthase). 2. Citrate -> isocitrate (aconitase). 3. Isocitrate -> alpha-ketoglutarate + CO2 + NADH (isocitrate dehydrogenase, main regulated step). 4. Alpha-ketoglutarate -> succinyl-CoA + CO2 + NADH (alpha-ketoglutarate dehydrogenase). 5. Succinyl-CoA -> succinate + GTP (succinyl-CoA synthetase). 6. Succinate -> fumarate + FADH2 (succinate dehydrogenase, also complex II). 7. Fumarate -> malate -> oxaloacetate + NADH. Per turn: 3 NADH, 1 FADH2, 1 GTP and 2 CO2. The cycle is amphibolic: it supplies intermediates for biosynthesis (citrate for fatty acids, oxaloacetate for glucose, alpha-ketoglutarate for amino acids). Regulation: ATP and NADH inhibit, ADP and calcium activate, at citrate synthase, isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase.

Complexes in the inner mitochondrial membrane - Complex I (NADH dehydrogenase): pumps 4 H+. - Complex II (succinate dehydrogenase): feeds FADH2 electrons, no proton pumping. - Coenzyme Q (ubiquinone) carries electrons to complex III (pumps 4 H+ ), cytochrome c carries them to complex IV (cytochrome oxidase, pumps 2 H+), which reduces O2 to water. Chemiosmotic model (Mitchell): the proton gradient (proton-motive force) is the energy store. ATP synthase (F0 channel and F1 catalytic head) makes ATP as protons flow back. Yield: about 2.5 ATP per NADH and 1.5 per FADH2. Complete oxidation of one glucose gives about 30-32 ATP. Regulation: respiratory control, meaning that ADP availability limits the rate. Uncouplers (2,4-dinitrophenol, thermogenin in brown fat) dissipate the gradient and produce heat. Inhibitors: rotenone (I), antimycin A (III), cyanide and carbon monoxide (IV), oligomycin (ATP synthase). Clinical tip: cyanide poisoning blocks cytochrome oxidase, and cells die of energy failure despite adequate oxygen.

1. Why does lactate form in an exercising muscle? 2. List the cofactors of pyruvate dehydrogenase. 3. Give the products of one turn of the Krebs cycle. 4. What is the proton-motive force? 5. Which complex does not pump protons? 6. How does an uncoupler affect ATP synthesis and heat?

1. To regenerate NAD+ so glycolysis can continue when oxygen supply is limited. 2. Thiamine pyrophosphate, lipoate, CoA, FAD and NAD+. 3. 3 NADH, 1 FADH2, 1 GTP, 2 CO2. 4. The electrochemical proton gradient across the inner mitochondrial membrane that drives ATP synthase. 5. Complex II. 6. It dissipates the gradient, so less ATP is made and more energy is released as heat.

PRPP (phosphoribosyl pyrophosphate) is made from ribose 5-phosphate. Purines: built atom by atom on PRPP, giving inosine monophosphate (IMP), which branches to AMP and GMP. Precursors: glycine, glutamine, aspartate, formate (from tetrahydrofolate) and CO2. The committed step is PRPP amidotransferase, inhibited by AMP, GMP and IMP. Pyrimidines: carbamoyl phosphate synthetase II (cytosol) and aspartate give orotate, which is attached to PRPP to make UMP. UMP gives UTP and CTP. Regulation is at carbamoyl phosphate synthetase II, inhibited by UTP. Deoxyribonucleotides: ribonucleotide reductase converts ribonucleoside diphosphates to deoxy forms. Thymidylate synthase (using methylenetetrahydrofolate) converts dUMP to dTMP. Clinical tip: hereditary orotic aciduria is a defect of UMP synthesis.

- Methotrexate inhibits dihydrofolate reductase and so depletes the folate needed for thymidylate and purines. - 5-Fluorouracil inhibits thymidylate synthase. - Hydroxyurea inhibits ribonucleotide reductase. - Mycophenolate inhibits IMP dehydrogenase, limiting guanine nucleotides in lymphocytes. - Azathioprine and 6-mercaptopurine are purine analogues.

Salvage: HGPRT converts hypoxanthine and guanine back to IMP and GMP using PRPP. APRT does the same for adenine. Complete HGPRT deficiency (Lesch-Nyhan syndrome) causes hyperuricaemia, self-mutilation and intellectual disability. Degradation - Purines are broken down to hypoxanthine, xanthine and finally uric acid by xanthine oxidase. Excess uric acid crystals cause gout. Allopurinol inhibits xanthine oxidase. - Pyrimidines are broken down to soluble products (beta-alanine, beta-aminoisobutyrate, CO2, ammonia). Clinical tip: adenosine deaminase deficiency causes severe combined immunodeficiency.

1. What is PRPP and which pathways use it? 2. Which enzyme catalyses the committed step of purine synthesis and what inhibits it? 3. How does methotrexate stop cell division? 4. What is the enzyme defect in Lesch-Nyhan syndrome? 5. What is the final product of human purine degradation? How does allopurinol act?

1. Phosphoribosyl pyrophosphate, made from ribose 5-phosphate. It is used in purine and pyrimidine de novo synthesis and in salvage. 2. PRPP amidotransferase, inhibited by AMP, GMP and IMP. 3. It inhibits dihydrofolate reductase, so the cell lacks tetrahydrofolate for thymidylate and purine synthesis. 4. HGPRT deficiency. 5. Uric acid. Allopurinol inhibits xanthine oxidase.

Classes: peptide and protein hormones (insulin, glucagon, growth hormone), amino-acid derivatives (adrenaline, noradrenaline, thyroxine, melatonin) and steroids. Peptide hormones are made as prohormones, stored in vesicles and released by exocytosis. They bind membrane receptors. - G-protein-coupled receptors: adenylate cyclase (cAMP, protein kinase A) or phospholipase C (IP3, diacylglycerol, calcium). - Receptor tyrosine kinases: insulin and growth factors. Insulin: made by pancreatic beta cells, lowers blood glucose (GLUT4 recruitment, glycogen and fat synthesis). Glucagon (alpha cells) raises it. Adrenaline mobilises glucose and fatty acids in stress. Clinical tip: in diabetes mellitus insulin is lacking (type 1) or tissues are resistant (type 2), giving hyperglycaemia.

Synthesis: from cholesterol in the adrenal cortex (cortisol, aldosterone), gonads (oestrogens, progesterone, testosterone). Not stored, made on demand. Transport: bound to carrier proteins in plasma (for example cortisol-binding globulin, sex hormone-binding globulin). Only the free fraction is active. Action: the hormone crosses the membrane and binds an intracellular receptor, which acts on DNA response elements to change gene transcription. Thyroid hormones behave similarly, although they are derived from tyrosine.

1. Give one example each of a peptide, amino-acid-derived and steroid hormone. 2. What second messengers do G-protein-coupled receptors use? 3. Why do steroid hormones act more slowly than peptide hormones? 4. Which hormones raise and lower blood glucose?

1. For example insulin, adrenaline, cortisol. 2. Cyclic AMP, or IP3 and diacylglycerol with calcium. 3. They change gene transcription through intracellular receptors. 4. Glucagon, adrenaline and cortisol raise it. Insulin lowers it.

1. Stomach: HCl denatures protein and activates pepsinogen to pepsin (an endopeptidase). 2. Pancreas: trypsinogen is activated by enteropeptidase in the duodenum to trypsin, which activates chymotrypsinogen, proelastase and procarboxypeptidases. Zymogens protect the pancreas from self-digestion. 3. Brush border: aminopeptidases and dipeptidases. Amino acids and di- and tripeptides are absorbed by specific transporters and reach the liver by the portal vein. Fate of amino acids: protein synthesis, energy (glucogenic and ketogenic amino acids) and precursors of glutathione, creatine, porphyrins (haem), catecholamines, histamine, serotonin, melanin, carnitine and nitric oxide. Clinical tip: acute pancreatitis involves premature activation of pancreatic zymogens.

Digestion: salivary and pancreatic alpha-amylase produce maltose and dextrins, and brush-border enzymes (maltase, sucrase-isomaltase, lactase) give glucose, galactose and fructose. Glucose and galactose enter by SGLT1, fructose by GLUT5. Glycaemic index (GI): the rise in blood glucose after a food compared with the same amount of carbohydrate as glucose. Glycaemic load also takes into account the amount eaten. Dietary fibre: soluble (pectins, beta-glucans, inulin), which lowers cholesterol and slows glucose absorption, and insoluble (cellulose), which speeds transit. Colonic bacteria ferment fibre to short-chain fatty acids such as butyrate, which nourish colonocytes. Prebiotics (inulin) promote beneficial bacteria.

Sources: oils, animal fat, dairy, fish, nuts. Types - Saturated fats raise LDL cholesterol. - Monounsaturated fats (olive oil) are neutral or beneficial. - Polyunsaturated omega-3 (fish, EPA and DHA) reduce triglycerides and are important for nerve tissue. Omega-6 (linoleic) is essential but should be balanced with omega-3. - Trans fatty acids (from partial hydrogenation) raise LDL and lower HDL. Smoke point matters in cooking: heating oils beyond it produces acrolein and oxidised lipids. Mediterranean diet: rich in olive oil, vegetables, fish and whole grains, and associated with lower cardiovascular risk.

1. Why are pancreatic proteases secreted as zymogens? 2. How are glucose and fructose absorbed? 3. Define glycaemic index and glycaemic load. 4. Compare soluble and insoluble fibre. 5. Which type of dietary fat is most harmful to blood lipids?

1. To prevent digestion of the pancreas itself; they are activated in the duodenum. 2. Glucose by SGLT1 (with sodium), fructose by GLUT5. 3. GI: blood glucose response relative to glucose. Glycaemic load: GI multiplied by the amount of carbohydrate eaten. 4. Soluble fibre slows glucose absorption, lowers cholesterol and is fermented. Insoluble fibre speeds transit. 5. Trans fats.