The Definitions Bank

• Numerically it is the pH midway between the pKa values of the ionisations on either side of the isoelectric species. For alanine (pKa 2.35 and 9.69) the pI is about 6.0.
• Above its pI a molecule carries a net negative charge and migrates to the anode; below its pI it is net positive and migrates to the cathode.

The basis of electrophoresis, isoelectric focusing and isoelectric precipitation. Acidic amino acids (Asp, Glu) have a low pI; basic ones (Lys, Arg, His) a high pI.

• Amino acids exist as zwitterions in blood and most tissues — the carboxyl group as –COO⁻ and the amino group as –NH₃⁺.
• The fully uncharged structure cannot exist in aqueous solution, because there is no pH at which the carboxyl group is protonated and the amino group is not.

Explains why amino acids are highly water-soluble crystalline solids with very high melting points, and why they act as buffers.

• It possesses partial double-bond character, so the peptide unit is rigid and planar and normally in the trans configuration.
• Rotation in the backbone is therefore restricted to the N–Cα bond (φ, phi) and the Cα–C bond (ψ, psi).

That restriction is what makes reproducible folding possible — the α-helix and β-sheet are simply the permitted combinations of φ and ψ.

• Harper's counts ten nutritionally essential L-α-amino acids.

The basis of protein quality in nutrition, and of the amino-acid requirements in parenteral feeding.

• Present in proteins from every domain of life; humans have about two dozen selenoproteins, including certain peroxidases and reductases and the iodothyronine deiodinases that convert thyroxine (T₄) to T₃.
• Incorporated co-translationally at a recoded UGA codon rather than having a codon of its own.

⚠️ Your slide says the genetic code specifies 20 amino acids; Harper's calls selenocysteine the 21st. Answer “20” if asked how many, but mention selenocysteine — both are right at different levels of detail.

• The sole exception is glycine, whose R group is a hydrogen atom, so the α-carbon carries two identical substituents and is not chiral.
• Only L-isomers occur in proteins, although free D-serine and D-aspartate exist in brain tissue.

The structural basis of enzyme stereospecificity — enzymes act on L-amino acids but not D, and on D-sugars but not L.

• It is determined by the nucleotide sequence of the gene, and dictates every higher order of structure and therefore the protein's function.

Clinically it serves as a molecular fingerprint identifying the protein, and as a route back to the gene that encodes it. Sickle cell disease shows how one residue can produce a disease.

• Proteins whose Stokes radius is too large to enter the pores are excluded, remain in the flowing mobile phase and elute first.
• Smaller proteins enter the pores, are retarded, and elute later — so proteins emerge in descending order of Stokes radius.

This is Practical Exp 1. Note the counter-intuitive elution order: large first. Writing “separates by molecular weight” loses the mark that “Stokes radius” earns.

• It is a function of both molecular mass and shape: a rapidly tumbling elongated protein sweeps out a larger effective volume than a spherical protein of the same mass, and so behaves as the larger molecule.

Why gel filtration is defined on Stokes radius rather than on mass alone.

• In theory only proteins that interact with the immobilised ligand adhere; all others wash through.
• Bound protein is eluted by competition with free soluble ligand, or less selectively with urea, guanidine hydrochloride, mildly acidic pH or high salt.
• Recombinant examples: a Ni²⁺ matrix binding a polyhistidine tag, a glutathione matrix binding a GST fusion.

The most selective single purification step available, because it separates on biological specificity rather than a bulk physical property.

• SDS binds at about one molecule per two peptide bonds, denaturing the polypeptide and conferring an approximately uniform charge-to-mass ratio — so charge is eliminated as a variable.
• Used with 2-mercaptoethanol or dithiothreitol to reduce disulfide bonds, it resolves the individual subunits of a multimeric protein.
• Bands are visualised with a dye such as Coomassie Blue.

The standard method for assessing protein purity.

• Each protein migrates until it reaches the region where the pH equals its isoelectric point (pI) — the pH at which its net charge is zero — and there it stops.
• The method is self-sharpening: a molecule diffusing either way regains charge and is driven back.

Combined with SDS-PAGE it gives two-dimensional electrophoresis, separating by pI in one dimension and M_r in the other — the basis of first-generation proteomics.

• Treatment with acid in a non-hydroxylic solvent then releases a phenylthiohydantoin (PTH) amino acid, identified by its chromatographic mobility, together with a peptide one residue shorter.
• Because the remaining peptide bonds are untouched, the process can be repeated on the newly exposed N-terminus — typically for 5–30 cycles.

The non-hydroxylic solvent is the whole trick: water would let the acid hydrolyse internal peptide bonds and destroy the chain.

• The first spectrometer separates individual peptides by mass and directs one selected peptide into the second, where it is fragmented and the masses of the fragments determined.

Used clinically to screen newborn blood for amino acids, fatty acids and other metabolites — diagnostic of phenylketonuria, ethylmalonic encephalopathy and glutaric acidaemia type 1. This is the biochemistry behind the heel-prick test.

• Unlike the genome it is not fixed: genes are switched on and off, different cell types express different proteins, expression changes with growth, differentiation and external stimuli, and many proteins are modified post-translationally.
• It is therefore described as a moving target.

Proteomics aims to identify proteins whose expression correlates with medically significant events. Note the two qualifiers that earn the marks: “an individual cell” and “at a particular time”.

• Different domains work together to provide the complete function of the protein.
• A small polypeptide such as triose phosphate isomerase or myoglobin may consist of a single domain; protein kinases contain two — a β-sheet-rich amino-terminal domain that binds ATP and an α-helix-rich carboxyl-terminal domain that binds the substrate.

Set in the 2020/21 paper. Do not confuse with a motif (short conserved region) or with supersecondary structure (10–40 residues, intermediate between secondary and tertiary).

• It arises when a series of consecutive aminoacyl residues adopt similar φ and ψ angles.
• The two commonest forms are the α-helix and the β-pleated sheet, together with bends, turns and loops.
• Stabilised principally by hydrogen bonds between backbone carbonyl oxygens and amide hydrogens.

Side chains are not involved in forming secondary structure, though they help determine its stability and type. That sentence is worth stating explicitly.

• A complete turn contains an average of 3.6 residues and rises 0.54 nm (the pitch).
• The R groups face outward; because proteins contain only L-amino acids, only right-handed α-helices occur.
• Stability arises from hydrogen bonds parallel to the helix axis, between the carbonyl oxygen of one peptide bond and the amide hydrogen of the fourth residue down the chain.

Broken by proline (its peptide nitrogen has no hydrogen to donate) and by glycine (too small and flexible).

• Monomeric proteins consist of one chain and have no quaternary structure.
• Greek letters distinguish subunit types and subscripts their number: α₄ is a homotetramer, α₂β₂ (adult haemoglobin) a heterotetramer.
• Stabilised by the same non-covalent forces as tertiary structure, and sometimes by interchain disulfide bonds.

The single structural difference between myoglobin and haemoglobin — and the source of every functional difference between them.

• The zinc finger: about 30 residues forming an elongated loop held at its base by a single Zn²⁺ ion coordinated to four residues — four Cys, or two Cys and two His.
• Other examples: nuclear localisation sequences, the Rossmann fold that binds NAD(P)H, EF hands that bind Ca²⁺.

Motifs are what bioinformatics algorithms look for — find the motif and you have a strong hypothesis about a new protein's function before running any assay.

• The commonest are combinations of α-helix and β-sheet.
• The helix-loop-helix motif, which provides the oligonucleotide-binding portion of DNA-binding proteins such as repressors and transcription factors, is the standard example.

Distinguish carefully from domain — a domain folds independently and performs a task; a supersecondary structure is a recurring structural theme.

• Within it, the modules of secondary structure rearrange until the mature, native conformation is attained.
• The process is orderly but not rigid — considerable flexibility exists in the order in which elements may be rearranged.

It is the answer to the paradox that a polypeptide with ≥10⁵⁰ possible conformations folds in milliseconds: folding is modular, not a random search.

• Prion diseases are protein conformation diseases: the pathological isoform PrP^Sc acts as a template converting the host's normal PrP^c — monomeric and α-helix rich — into the β-sheet-rich PrP^Sc, which aggregates into insoluble, protease-resistant deposits.
• The amino acid sequence is unchanged; only the conformation differs.

Prion disease can manifest as infectious, genetic or sporadic — the reason no viral or bacterial gene could ever be found.

• Configuration is the geometric relationship between a given set of atoms — for example that which distinguishes L- from D-amino acids — and its interconversion requires breaking covalent bonds.

The one-line test: do you have to break a covalent bond to get from one to the other? If yes, configuration. If no, conformation.

• The β-position substituents are methyl, vinyl and propionate.
• The iron forms six coordination bonds: four to the pyrrole nitrogens, a fifth to the proximal histidine F8, and a sixth reserved for oxygen or left unoccupied.
• Its network of conjugated double bonds absorbs visible light and colours haem deep red.

Oxidation of Fe²⁺ to Fe³⁺ destroys biological activity — the opposite of the cytochromes, where redox cycling of the metal is essential.

• Some proteins consist of polypeptide alone; others, such as myoglobin and haemoglobin, require a prosthetic group — in their case haem, held in a hydrophobic pocket by non-covalent bonding.
• In enzymes, prosthetic groups are tightly and stably incorporated by covalent or non-covalent forces; metal ions are the commonest type.

Distinguish from a cofactor (binds reversibly, must be present in the medium) and a coenzyme (a recyclable substrate shuttle).

• It permits haemoglobin to maximise both the quantity of O₂ loaded at the pO₂ of the lungs and the quantity released at the pO₂ of the peripheral tissues.
• It is the reason the dissociation curve is sigmoid rather than hyperbolic.

Cooperative interactions are an exclusive property of multimeric proteins — which is why monomeric myoglobin cannot show cooperativity, a sigmoid curve, or the Bohr effect.

• A high P₅₀ indicates LOW affinity, and therefore better release of oxygen at the tissues. In all instances P₅₀ exceeds the pO₂ of the peripheral tissues.
• HbA: 26 mm Hg. HbF: 20 mm Hg.

HbF's lower P₅₀ lets it extract oxygen from maternal HbA across the placenta — but the same high affinity limits delivery to the tissues after birth.

• The transition is triggered when O₂ binding pulls the haem iron into the plane of the ring, moving His F8, rupturing the salt bridges and rotating one α/β pair through 15°.
• It does not occur at a fixed number of bound O₂ — it becomes progressively more probable with each successive oxygen.

The same terms describe the low- and high-affinity conformations of allosteric enzymes (Unit 7). Everything that stabilises T — protons, CO₂, chloride, BPG — lowers affinity and improves tissue delivery.

• In the tissues: CO₂ forms carbonic acid, which dissociates to bicarbonate and protons. Deoxyhaemoglobin binds one proton for every two O₂ released, acting as a buffer; the lower pH stabilises the T state and enhances O₂ delivery.
• In the lungs: oxygen uptake releases protons, which combine with bicarbonate to form carbonic acid; carbonic anhydrase dehydrates this to CO₂, which is exhaled. Oxygen binding therefore drives CO₂ exhalation.
• The protons arise from rupture of salt bridges involving β-chain His 146.

The effect depends on cooperativity and is therefore absent in myoglobin.

• Of the over 1100 known mutations affecting human haemoglobins, almost all are extremely rare and benign and present no clinical abnormality; the term is reserved for those that impair function.
• More than 7% of the world's population carry a haemoglobin disorder.

⚠️ Your slide gives “over 800” mutations, Harper's “over 1100” — an edition difference. Quote the lecturer's figure.

• Normally the enzyme methaemoglobin reductase reduces Fe³⁺ back to Fe²⁺.
• Methaemoglobinaemia arises from oxidation by agents such as sulfonamides, from hereditary haemoglobin M (in which His F8 is replaced by tyrosine, whose phenolate anion stabilises Fe³⁺), or from reduced methaemoglobin reductase activity.

Contrast HbS, where a surface mutation leaves oxygen binding intact but makes the protein sticky, with HbM, where a mutation at the iron-bonded residue abolishes binding outright.

• Enzymes are additionally stereospecific, typically catalysing the reaction of only one stereoisomer — D- but not L-sugars, L- but not D-amino acids.
• Stereospecificity arises from binding at at least three points of attachment, which fixes the substrate's orientation so that apparently identical groups become distinguishable.

Specificity is what allows a cell to conduct and independently control a broad spectrum of biochemical processes simultaneously in the same small volume.

• It comprises only a small part of the molecule.
• The catalytic residues are far apart in the primary structure and are brought together only by the three-dimensional fold.
• It lies in a cleft or crevice, shielding the substrate from water, and provides a microenvironment whose polarity, hydrophobicity, acidity or alkalinity may differ markedly from the cytoplasm.

A TMU study question asks for the characteristics, not just the definition — so give the four points above. The residues being far apart in sequence is the fact most often tested.

• Most cofactors are metal ions; enzymes requiring one are called metal-activated enzymes, as distinct from the metalloenzymes in which a tightly bound metal serves as a prosthetic group.

The distinction from a prosthetic group is entirely one of tightness of binding.

• Its role is twofold: to stabilise reactive species such as hydride ions (NADH) or hydrogen atoms (FADH), which are too reactive to persist freely; and to act as an adaptor or handle facilitating recognition of small groups such as acetate (CoA) or glucose (UDP) by target enzymes.
• Many are derivatives of B vitamins — nicotinamide → NAD⁺/NADP⁺, riboflavin → FMN/FAD, pantothenic acid → coenzyme A, thiamin → thiamin pyrophosphate.

Coenzymes, cofactors and prosthetic groups all exist to extend catalytic capability beyond the limited functional groups of amino acid side chains. ⚠️ Not every enzyme requires one.

• Although the reaction is identical, their physical, chemical and immunological properties differ significantly — which is what allows them to be separated by electrophoresis.
• Lactate dehydrogenase is a tetramer of H (heart) and M (muscle) subunits, giving five isozymes; LDH-1 predominates in heart, LDH-5 in liver.

Catalysing the same reaction is the only similarity — a favourite MCQ. Clinically, the plasma isozyme pattern reveals which organ has been damaged.

• Substrate and enzyme induce mutual conformational changes that facilitate both substrate recognition and catalysis.
• It replaced Emil Fischer's “lock and key” model, which pictured a rigid complementary site and failed to account for the dynamic changes that accompany catalysis.

Not merely a nicer picture: a rigid site could hold a substrate but never distort it, whereas a site that closes around its substrate can strain the bond destined for cleavage.

• Proteins that diverged from a common ancestor and share a large number of conserved residues are said to be homologous.
• Among the most highly conserved residues are those that participate directly in catalysis.

Evolution tolerates change almost anywhere except the active site — which is why sequence comparison can identify an unknown enzyme's mechanism.

• With the exception of the ribozymes, the vast majority of enzymes are proteins.

This is why the safe formulation is “enzymes are mostly protein catalysts”. A statement that all enzymes are proteins is false, and that is a frequent MCQ.

• It has the dimensions of substrate concentration, not of rate.
• It varies inversely with affinity: a low K_m means high affinity.
• It is independent of enzyme concentration (which affects V_max instead), but is affected by the nature of the enzyme and substrate, by temperature and by pH.
• A competitive inhibitor raises the apparent K_m; a non-competitive inhibitor leaves it unchanged.

Physiologically: hexokinase has a low K_m and works even at low blood glucose, whereas hepatic glucokinase has a high K_m and acts only after a meal. Same reaction, two K_m values, two roles.

• When [S] ≪ K_m, v_i is directly proportional to [S].
• When [S] = K_m, v_i = V_max/2 — which defines K_m.
• When [S] ≫ K_m, v_i ≈ V_max and is unaffected by further substrate.

For any fraction f of V_max, [S] = K_m × f/(1−f). So 80% needs 4 K_m, 90% needs 9, and 99% needs 99 — which is why Lineweaver–Burk exists.

• It is reached when the enzyme is saturated — when all the enzyme is present as the ES complex and no free enzyme remains available to bind further substrate.

Memorise the phrase “no free enzyme remains available”; it is what earns the mark when asked to explain saturation kinetics.

• It acts by decreasing the number of free enzyme molecules available to form ES, and is overcome by raising [S].
• Kinetics: apparent K_m INCREASED, V_max UNCHANGED. On a double-reciprocal plot the lines converge on the y-axis.

Classic example: malonate inhibiting succinate dehydrogenase — it binds the active site but has only one methylene carbon, so it cannot be dehydrogenated. The statins are the clinical example.

• Because inhibitor binding does not affect substrate binding, K_m is unchanged; but the EI complex, while it can still bind substrate, is less efficient at transforming it into product.
• Kinetics: K_m UNCHANGED, V_max DECREASED. The inhibition cannot be overcome by excess substrate, and the lines converge on the x-axis.

Raising [S] only helps when the problem is access to the site. Here it never was.

• Because these covalent changes are relatively stable, an enzyme “poisoned” by a heavy metal atom or an acylating reagent remains inhibited even after the inhibitor is removed from the surrounding medium.

The key contrast with reversible inhibitors, from which fully active enzyme is recovered simply by washing the inhibitor away. Suicide inhibitors are substrate analogues that the enzyme's own machinery converts into an irreversible poison.

• IC₅₀ is a less rigorous measure, because unlike an equilibrium constant its numerical value varies with the conditions — substrate concentration and so on — under which it is determined.

K_i is calculable from the x-intercept of a Lineweaver–Burk plot once K_m is known, or from a Dixon plot.

• A ratio is used because the benefit of a high k_cat can only be realised if K_m is sufficiently low — processing speed is worthless if the enzyme cannot capture substrate at physiological concentrations.
• Enzymes for which formation of the ES complex is rate-limiting are termed diffusion-limited or “catalytically perfect”.

Related measures: specific activity = V_max ÷ protein concentration (impure preparations); turnover number = V_max ÷ moles of enzyme.

• Raising temperature increases both the kinetic energy of molecules — so more exceed the activation-energy barrier — and the frequency of collisions.
• This holds only over a limited range: continued heating causes the enzyme to denature, so every enzyme has a temperature optimum.

• Enzymes are not changed by the reaction they catalyse; they do not change the equilibrium, and so cannot force an energetically unfavourable reaction; they increase rates by decreasing the activation energy.

• Allosteric enzymes are accordingly those whose catalysis at the active site may be modulated by the presence of effectors at an allosteric site.
• Jacques Monod reasoned that because most feedback inhibitors bear no structural similarity to the substrate, they are not isosteric but allosteric — “occupying another space”.
• K-series enzymes: the inhibitor raises K_m without affecting V_max. V-series: it lowers V_max without affecting K_m.

Set in the 2019 paper. Most feedback inhibition works this way, and many hormones act through allosteric second messengers.

• Reversible modifications include phosphorylation (by far the commonest), acetylation, methylation and ADP-ribosylation. Protein kinases transfer the terminal γ-phosphoryl group of ATP to seryl, threonyl or tyrosyl residues; protein phosphatases remove it hydrolytically.
• Irreversible modification is partial (selective) proteolysis, as in the activation of zymogens.
• Both are short-term regulation, acting within seconds, unlike induction of protein synthesis which takes hours.

Set in the 2020/21 paper. Note the subtlety: “reversible” means the protein can be restored, not that the same reaction runs backwards — removal uses a different, separately favourable reaction.

• In most cases it operates by allosteric regulation.
• In branched pathways, fine control is achieved by multiple feedback loops, by cooperative feedback inhibition (two end products acting additively or more than additively), or by multiple isoforms each sensitive to a different end product.

⚠️ Distinguish from feedback regulation, a phenomenological term devoid of mechanistic implications. Inhibiting an early step is the point — inhibiting the last enzyme would leave intermediates accumulating uselessly.

• Proteolysis produces conformational changes that properly configure the active site — in chymotrypsin it aligns the Asp102–His57–Ser195 catalytic triad.
• Examples: pepsinogen → pepsin, trypsinogen → trypsin, chymotrypsinogen → chymotrypsin, proinsulin → insulin, procollagen → collagen, and the clotting and complement factors.
• Physiologically irreversible, because cells cannot reunite the two portions of a protein produced by hydrolysis of a peptide bond.

Three reasons cells use them: protection of the tissue of origin from autodigestion (pancreatitis); rapid mobilisation on demand, since synthesis would be far too slow to respond to blood loss; and economy.

• By contrast, the concentrations of many enzymes depend on the presence of inducers — typically substrates or structurally related compounds — that initiate their synthesis.

Induction of protein synthesis requires hours, so changes in enzyme level serve long-term adaptive requirements rather than rapid change.

• Decreasing the quantity or catalytic efficiency of that enzyme immediately reduces flux through the entire pathway; increasing either enhances it.

Such enzymes are efficient drug targets: the statins inhibit HMG-CoA reductase, the rate-limiting enzyme of cholesterogenesis. Acetyl-CoA carboxylase plays the same role in fatty acid synthesis.

• It targets proteins to the interior of the 26S proteasome, a complex of more than 30 subunits forming a hollow cylinder whose proteolytic active sites face inward, preventing indiscriminate degradation.

Degrades both regulated proteins (the cyclins) and proteins damaged by loss of a prosthetic group or oxidation of residues. Its dysfunction contributes to the misfolded-protein accumulation of several neurodegenerative diseases.

• Examples include cAMP, 3′,5′-cGMP, Ca²⁺, nitric oxide and the polyphosphoinositols produced by hormone-regulated phospholipases.

The kinases and phosphatases responding to these signals constitute a “bio-organic computer” that integrates complex environmental information into a coherent cellular response.

• Achieved passively, because substrate concentrations sit near K_m where the curve is steepest, so flux tracks [S] automatically;
• and actively, by changing the quantity (hours) or catalytic efficiency (seconds) of the enzyme catalysing the committed, rate-limiting reaction.

A TMU study question asks “how is this achieved?” — give both the passive and the active levels, and name the two timescales.

• Biological systems are essentially isothermic and therefore use chemical energy, not heat, to power living processes — there is no temperature gradient to exploit, so energy must be moved around chemically.

This constraint is why a carrier molecule such as ATP has to exist at all.

• ΔG = ΔH − TΔS, combining the two laws of thermodynamics.
• Negative ΔG = EXERGONIC: proceeds spontaneously; if large, essentially irreversible. Positive = ENDERGONIC: needs energy supplied. Zero = equilibrium.
• ΔG⁰′ is the standard free-energy change at pH 7.0, related to the equilibrium constant by ΔG⁰′ = −RT ln K′_eq.

The sign rules govern every later pathway — they are what makes three steps of glycolysis irreversible and therefore regulable.

• The group comprises anhydrides (1,3-bisphosphoglycerate), enolphosphates (phosphoenolpyruvate) and phosphoguanidines (creatine phosphate, arginine phosphate).
• Low-energy phosphates — the ester phosphates of the glycolytic intermediates — fall below ATP.
• The symbol ~℗ denotes that transfer of the group yields the larger quantity of free energy; “group transfer potential” is preferred to “high-energy bond”.

ATP's intermediate position is the whole point: it can be regenerated by the compounds above it and can phosphorylate everything below.

• It turns over very rapidly, because the total ATP/ADP pool is sufficient to maintain an active tissue for only a few seconds.
• The three sources of ~℗: oxidative phosphorylation (the greatest quantitative source), glycolysis (net 2, at phosphoglycerate kinase and pyruvate kinase) and the citric acid cycle (1, at succinate thiokinase).

ATP is not a store of energy but a flow — made continuously at the rate it is spent, which is why interrupting the respiratory chain kills within minutes.

• Phosphagens permit ATP concentration to be maintained when it is rapidly utilised, and accumulate when the ATP/ADP ratio is high.

Creatine phosphate lies above ATP in the free-energy table (−43.1 kJ/mol), which is precisely what lets it rephosphorylate ADP instantly.

• Electrons pass down the respiratory chain in the inner mitochondrial membrane to their final reaction with oxygen to form water.
• The free energy released is used by Complexes I, III and IV, acting as proton pumps, to translocate H⁺ into the intermembrane space.
• The resulting proton motive force drives ATP synthase to phosphorylate ADP.

⭐ Set in BOTH readable papers — one of only two terms proven to repeat. It is also the greatest quantitative source of high-energy phosphate in aerobic organisms.

• The resulting proton motive force — an electrochemical potential difference, negative on the matrix side — drives ATP synthesis.
• Complexes I, III and IV act as proton pumps; because the inner membrane is impermeable to protons, they accumulate in the intermembrane space.

The insight was that there is no chemical intermediate linking oxidation to phosphorylation — the link is a gradient. It explains uncouplers, oligomycin and respiratory control in one idea.

• Uncouplers are toxic in vivo, causing respiration to become uncontrolled, since its rate is no longer limited by ADP or P_i; the energy is released as heat. The commonest is 2,4-dinitrophenol (DNP).

Contrast oligomycin, which blocks H⁺ conduction through F₀ and therefore stops both oxidation and phosphorylation. Uncoupler: oxidation accelerates. Oligomycin: both stop.

• It arises because oxidation and phosphorylation are tightly coupled: oxidation cannot proceed without concomitant phosphorylation of ADP.
• Most cells at rest are in state 4, ADP-limited.

The regulator is the substrate itself — demand automatically creates its own supply signal, with no allosteric machinery required.

• ⚠️ Your sources disagree. Lecture 9 slide 16 gives 3 (NADH) and 2 (FADH₂). Harper's p.131 gives 2.5 and 1.5.
• The rest of your course agrees with Harper's: Lecture 10 slide 13 states 2.5 and 1.5 explicitly; Lecture 11 slide 36 gives 32 ATP per glucose, only possible with those values; and Lecture 9 slide 27 itself concedes “possibly 2.5”.
• The values are non-integer because 10 H⁺ pumped per NADH ÷ about 4 H⁺ per ATP exported does not divide evenly.

Use 2.5 and 1.5. They match Harper's, the majority of your slides, and the 10-per-turn and 32-per-glucose figures. Explain with the proton count — worth more than either pair of numbers.

• Overall anaerobic equation: glucose + 2 ADP + 2 P_i → 2 lactate + 2 ATP + 2 H₂O.
• Regulated at three markedly exergonic, physiologically irreversible steps — hexokinase (and glucokinase), phosphofructokinase and pyruvate kinase.
• It is also the main pathway for the metabolism of fructose, galactose and other dietary carbohydrates.

Net 2 ATP anaerobically, up to 32 aerobically. Erythrocytes rely on it entirely, which is why glycolytic enzyme defects present as haemolytic anaemia.

• Hexokinase — most tissues, high affinity (low K_m), so saturated even at low blood glucose.
• Glucokinase — liver and pancreatic β islet cells, K_m very much higher, so active only when glucose is high.
• In liver it removes glucose from the blood after a meal, providing G6P in excess of glycolytic needs for glycogen synthesis and lipogenesis. In pancreas, the G6P formed signals glucose availability and leads to insulin secretion.

The clearest demonstration in the course that K_m is not an abstraction — same reaction, two values, two physiological roles.

• Three catalytic enzymes: pyruvate dehydrogenase, dihydrolipoamide transacetylase, dihydrolipoamide dehydrogenase.
• Five cofactors: TPP, lipoic acid, NAD⁺, FAD and CoASH — four of them B-vitamin derived.
• Regulated by end-product inhibition (acetyl-CoA, NADH) and by covalent modification — phosphorylation of three serine residues inactivates; the dephosphorylated form is active.

Its irreversibility is why fat cannot be converted to glucose. Its inhibition during starvation spares carbohydrate for the brain.

• It is the final common pathway for the aerobic oxidation of carbohydrate, lipid and protein, because glucose, fatty acids and most amino acids are metabolised to acetyl-CoA or to cycle intermediates.
• Located in the mitochondrial matrix. Acetyl-CoA condenses with oxaloacetate to form citrate; two CO₂ are released and oxaloacetate is regenerated, so it plays a catalytic role.
• One turn yields 10 ATP: 3 NADH × 2.5, 1 FADH₂ × 1.5, and 1 substrate-level ATP at succinate thiokinase.

⭐ Its significance in the metabolism of carbohydrate, triacylglycerol and amino acids was a Section II question in both readable papers.

• The citric acid cycle is not only a pathway for oxidation of two-carbon units; it is also a major pathway for the interconversion of metabolites arising from transamination and deamination, and it provides substrates for amino acid synthesis, gluconeogenesis and fatty acid synthesis.

The exit to gluconeogenesis is PEP carboxykinase (using GTP); the exit to lipogenesis is citrate, cleaved in the cytosol by ATP-citrate lyase.

• The most important is the carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase, maintaining an adequate concentration of oxaloacetate for condensation with acetyl-CoA.
• If acetyl-CoA accumulates it acts BOTH as an allosteric activator of pyruvate carboxylase AND as an inhibitor of pyruvate dehydrogenase — ensuring its own supply of oxaloacetate.

This is the mechanism behind “fat burns in the flame of carbohydrate”, and therefore behind starvation ketosis.

• Liver: up to 6%. Muscle: rarely over 1% — but because of its greater mass, muscle contains three to four times as much in total.
• Muscle glycogen supplies glycolysis within the muscle itself; liver glycogen stores and exports glucose to maintain blood glucose, and is almost totally depleted after 12–18 hours of fasting.

The difference is one enzyme: glucose-6-phosphatase is present in liver and kidney but not muscle, so only the liver can release free glucose to the blood.

• Glycogen phosphorylase catalyses the rate-limiting step, the phosphorolytic cleavage by inorganic phosphate of the 1→4 linkages to yield glucose 1-phosphate.
• Residues are removed until about four remain on either side of a 1→6 branch; a transferase moves a trisaccharide to expose the branch point, and debranching enzyme hydrolyses the 1→6 linkage.
• In liver and kidney only, glucose-6-phosphatase then yields free glucose for export.

Phosphorolysis, not hydrolysis — the product is already phosphorylated, saving the ATP hexokinase would otherwise spend.

• Von Gierke's disease (type I) — glucose-6-phosphatase deficiency: hepatic glycogen accumulates but cannot be exported, causing fasting hypoglycaemia.
• McArdle's syndrome (type V) — muscle glycogen phosphorylase deficiency: exercise intolerance and cramps, with no rise in blood lactate.

Two diseases, two tissues, two purposes — derivable from the liver/muscle distinction rather than memorised.

• Major substrates: the glucogenic amino acids, lactate, glycerol and propionate. Major tissues: liver and kidney.
• It uses the reversible glycolytic reactions plus four bypass enzymes that circumvent the three irreversible steps: pyruvate carboxylase (mitochondrial, ATP, biotin) and PEP carboxykinase (GTP); fructose 1,6-bisphosphatase; and glucose-6-phosphatase.
• It clears lactate produced by muscle and erythrocytes and glycerol produced by adipose tissue.

Acetyl-CoA is not a substrate — pyruvate dehydrogenase is irreversible, which is why fat cannot be converted to glucose and starvation eventually consumes muscle protein.

• It is formed by phosphorylation of fructose 6-phosphate by phosphofructokinase-2; the same enzyme protein is also responsible for its breakdown, since it has fructose-2,6-bisphosphatase activity — a bifunctional enzyme.
• Which activity dominates is set by cAMP-dependent phosphorylation, so glucagon lowers it and switches the liver to gluconeogenesis.

⚠️ It is a regulatory signal only, NOT a pathway intermediate — unlike fructose 1,6-bisphosphate. Examiners set this confusion deliberately.

• It is energetically expensive for the liver, which must supply the ATP and GTP.

The resulting rise in oxygen consumption is the oxygen debt after exercise, and the same mechanism underlies the hypermetabolism of cancer cachexia.

• Cytosolic. Oxidation is by dehydrogenation, but NADP⁺ and not NAD⁺ is the hydrogen acceptor, and CO₂ — not produced in glycolysis at all — is a characteristic product.
• An oxidative, non-reversible phase (G6PD, gluconolactone hydrolase, 6-phosphogluconate dehydrogenase → 2 NADPH + CO₂ + ribulose 5-phosphate) and a non-oxidative, reversible phase (mainly transketolase and transaldolase).
• It is called a shunt because it begins at glucose 6-phosphate and rejoins glycolysis at glyceraldehyde 3-phosphate and fructose 6-phosphate.

Three ways to recognise it in a question: NADP appears · CO₂ appears · no ATP appears.

• Mechanism: no G6PD → no NADPH → glutathione reductase (an FAD flavoprotein) cannot regenerate reduced glutathione → glutathione peroxidase (which contains selenocysteine at its active site) cannot remove H₂O₂ → oxidative damage to the cell membrane → haemolysis.
• The erythrocyte is uniquely vulnerable because it has no other source of NADPH and no nucleus with which to make more enzyme.

This is what Unit 1's “21st amino acid” is actually for — and why selenium is a dietary trace element.

• Classification: simple lipids (fats and waxes — esters of fatty acids with alcohols); complex lipids (phospholipids, glycolipids — esters containing an additional group); precursor and derived lipids (fatty acids, glycerol, steroids, ketone bodies, fat-soluble vitamins, hormones).
• Functions: the most concentrated form of energy storage (9 kcal/g against 4 for carbohydrate and protein); structural components of membranes; thermal insulation and mechanical protection; carriers of the fat-soluble vitamins A, D, E and K; and precursors of steroid hormones and eicosanoids.

The definition is a solubility definition, not a structural one — that is the point examiners look for. Say “related by physical rather than chemical properties” and the first mark is yours.

• Mammals possess Δ9, Δ6, Δ5 and Δ4 desaturases but no desaturase beyond carbon 9, so they cannot introduce a double bond in the ω-6 or ω-3 position.
• Arachidonic acid can be made from linoleic acid and is therefore essential only when linoleic acid is lacking. It is the precursor of the eicosanoids.
• Deficiency causes a scaly dermatitis with increased water loss through the skin, impaired growth and impaired wound healing.

The dietary requirement at one end of the chain determines the availability of prostaglandins, thromboxanes and leukotrienes at the other.

• Three classes: prostaglandins (PG), which have a cyclopentane ring; thromboxanes (TX), in which that ring contains an oxygen atom; and leukotrienes (LT), which have no ring but three conjugated double bonds.
• Two routes from arachidonic acid: the cyclooxygenase pathway gives prostaglandins and thromboxanes, the lipoxygenase pathway gives leukotrienes.
• Actions: PGs mediate inflammation, pain and fever and protect the gastric mucosa; TXA₂ causes platelet aggregation and vasoconstriction, PGI₂ (prostacyclin) does the opposite; leukotrienes cause bronchoconstriction and are important in asthma.

The pathway is the pharmacology. Aspirin and NSAIDs inhibit cyclooxygenase — which is why they are both anti-inflammatory and ulcerogenic; low-dose aspirin is antiplatelet through TXA₂; leukotriene antagonists treat asthma.

• In an aqueous environment they orient themselves so that the polar groups face the water and the hydrocarbon chains face each other, forming bilayers, micelles, liposomes and the surface monolayer of lipoproteins.
• This single property underlies the structure of every biological membrane, the emulsifying action of bile salts, and the solubility of lipoproteins in plasma.

One physical principle explains membranes, fat digestion and lipid transport at once — worth naming explicitly in any of those three answers.

• Acetyl-CoA is the immediate substrate and free palmitate is the end product. Reducing equivalents are supplied as NADPH, chiefly from the pentose phosphate pathway; the acyl carrier is acyl carrier protein (ACP).
• Overall: acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H⁺ → palmitate + 7 CO₂ + 6 H₂O + 8 CoA-SH + 14 NADP⁺.
• Acetyl-CoA reaches the cytosol as citrate, cleaved there by ATP-citrate lyase — the citrate shuttle.

Note that the tissues with active lipogenesis are exactly those with an active pentose phosphate pathway, and that both pathways are cytosolic so NADPH passes freely between them.

• It requires bicarbonate as the source of CO₂, ATP, and the B vitamin biotin, and acts in two steps: carboxylation of biotin involving ATP, then transfer of the carboxyl group to acetyl-CoA.
• Activated allosterically by citrate, which converts the inactive dimer into an active polymeric form of several million daltons; citrate rises in the well-fed state and is an indicator of a plentiful supply of acetyl-CoA.
• Inhibited by phosphorylation (glucagon, epinephrine) and by long-chain acyl-CoA — negative feedback by a product of the pathway.

Malonyl-CoA is also the inhibitor of carnitine palmitoyltransferase-I, so this one enzyme reciprocally regulates fatty acid synthesis and oxidation across two compartments.

• It is a homodimer of two identical subunits, each containing six enzymes and an ACP, arranged in an X shape. ACP contains the vitamin pantothenic acid as 4′-phosphopantetheine and has a function similar to CoA in β-oxidation.
• Two advantages of the multienzyme arrangement: it achieves the effect of compartmentalisation without the erection of permeability barriers, since the intermediates never leave the complex; and synthesis of all the enzymes is coordinated, being encoded by a single gene.
• The priming acetyl-CoA forms carbons 15 and 16 of palmitate; all subsequent C₂ units come from malonyl-CoA. Thioesterase releases free palmitate.

Propionyl-CoA primes the synthesis of odd-chain fatty acids, found particularly in ruminant fat and milk.

• The fatty acid is first activated to acyl-CoA by acyl-CoA synthetase at the cost of two high-energy phosphates (ATP → AMP + PPi), then carried into the matrix by the carnitine shuttle.
• Each cycle comprises oxidation (FAD) → hydration → oxidation (NAD⁺) → thiolytic cleavage, yielding one acetyl-CoA, one FADH₂, one NADH and an acyl-CoA two carbons shorter — 4 ATP per cycle from the reducing equivalents alone.
• Complete oxidation of palmitate yields 106 ATP net (7 cycles: 7 FADH₂ + 7 NADH + 8 acetyl-CoA, less 2 for activation).
• Odd-chain acids end with propionyl-CoA, converted via methylmalonyl-CoA (biotin) to succinyl-CoA (vitamin B₁₂).

Fatty acids are not glucogenic, because pyruvate dehydrogenase is irreversible — the propionyl-CoA of odd-chain acids is the sole exception. A frequently examined negative fact.

• Carnitine (β-hydroxy-γ-trimethylammonium butyrate) is widely distributed and particularly abundant in muscle.
• Three components in sequence: CPT-I on the outer membrane forms acylcarnitine; the carnitine-acylcarnitine translocase exchanges it inwards for free carnitine; CPT-II on the inner surface regenerates acyl-CoA in the matrix. Carnitine is returned, so it is a catalytic carrier, not a consumed substrate.
• CPT-I is the rate-limiting step of fatty acid oxidation and is inhibited by malonyl-CoA.
• Carnitine deficiency causes hypoglycemia, hypoketonemia, and lipid accumulation with muscular weakness; it responds to oral carnitine.

Do not confuse it with the citrate shuttle, which carries acetyl units OUT for fatty acid synthesis. Carnitine carries acyl groups IN.

• The liver is the only organ that adds significant quantities of ketone bodies to the blood; extrahepatic tissues consume them. The liver cannot use them, because it lacks succinyl-CoA-acetoacetate CoA transferase.
• 3-hydroxybutyrate is quantitatively predominant in blood and urine in ketosis; the acetoacetate/3-hydroxybutyrate ratio depends on the mitochondrial [NAD⁺]/[NADH] ratio. Acetone is not metabolised and is exhaled, giving the ketotic breath.
• They are normal fuels of respiration, important for heart muscle and renal cortex, and cross the blood-brain barrier, which albumin-bound free fatty acids cannot — so the brain uses them in prolonged starvation, sparing body protein.
• Formation: 2 acetyl-CoA → acetoacetyl-CoA → (HMG-CoA synthase, the regulatory enzyme) → HMG-CoA → (HMG-CoA lyase) → acetoacetate.

Ketogenesis is the overflow route for acetyl-CoA the citric acid cycle cannot accept, because oxaloacetate has been drawn into gluconeogenesis — “fat burns in the flame of carbohydrate.”

• It manifests as ketonemia, ketonuria and a ketotic (acetone) breath.
• Mild ketosis occurs normally in starvation, after exercise and in pregnancy; it is severe in diabetes mellitus, where insulin lack removes the restraint on lipolysis. Since acetoacetate and 3-hydroxybutyrate are relatively strong acids, the result is ketoacidosis.
• In ruminants it is a serious problem in lactating cattle and pregnant ewes.

Ketosis is a matter of rate, not of a defective pathway — state that and the definition is already half made.

• Structure: a nonpolar core of triacylglycerol and cholesteryl ester, surrounded by a surface monolayer of amphipathic phospholipid, free cholesterol and apolipoprotein, polar faces outward to the plasma.
• Four major classes, in order of increasing density and decreasing size: chylomicrons (intestine; dietary triacylglycerol; apo B-48); VLDL (liver; endogenous triacylglycerol; apo B-100); LDL (from VLDL; cholesterol-rich; delivers cholesterol to tissues); HDL (reverse cholesterol transport; apo A-I; highest protein content, about 50%).
• Since protein is denser than lipid, the more lipid a particle carries the larger and the less dense it is — density and diameter run in opposite directions.

LDL correlates positively and HDL inversely with the incidence of atherosclerosis, because the two carry cholesterol in opposite directions.

• Three functions: structural — they hold the particle together and confer solubility; enzyme cofactors; and ligands for cell-surface receptors, determining where the particle is delivered.
• apo B-100 — liver; VLDL, IDL, LDL; the ligand for the LDL receptor. apo B-48 — intestine; chylomicrons; 48% of the length of B-100, from the same gene by RNA editing, and lacking the receptor-binding domain.
• apo A-I — HDL; activates LCAT. apo C-II — activates lipoprotein lipase. apo E — mediates hepatic uptake of chylomicron remnants and IDL.

Because apo B-48 lacks the receptor-binding domain, chylomicron remnants must be cleared through apo E instead — the two facts belong together in an answer.

• It requires apo C-II as an activator and phospholipid as a cofactor, so a lipoprotein effectively licenses its own digestion by carrying the right apolipoprotein.
• Insulin induces its synthesis in adipose tissue, directing fat to storage in the fed state.
• Do not confuse with hormone-sensitive lipase, which is intracellular, releases fatty acids out of the adipocyte, is activated by phosphorylation in response to epinephrine and glucagon, and is inhibited by insulin — the principal antilipolytic hormone.
• Deficiency causes familial hypertriacylglycerolemia (type I) with chylomicronemia.

Two lipases, opposite jobs, opposite hormonal control — the single most common confusion in this unit.

• Nascent discoidal HDL takes up free cholesterol from cell membranes via the transporter ABCA1.
• LCAT, activated by apo A-I, esterifies it using a fatty acid from phosphatidylcholine; the nonpolar ester moves into the core, keeping the surface concentration low so that more cholesterol can flow in, and the disc becomes spherical HDL₃ then HDL₂.
• The cholesteryl ester returns to the liver directly via scavenger receptor B1 or indirectly, transferred by CETP to VLDL and LDL.
• ABCA1 deficiency = Tangier disease, with near-absent HDL.

This is the mechanistic reason for the inverse relationship between HDL concentration and coronary risk.

• Category 1 — too much fat arriving: raised plasma free fatty acids from adipose tissue, as in starvation, diabetes mellitus and high-fat diets; accompanied by ketosis.
• Category 2 — a metabolic block in VLDL production: a block in apolipoprotein synthesis (abetalipoproteinemia; carbon tetrachloride, chloroform, phosphorus, lead, arsenic, puromycin; protein deficiency); a failure to provide phospholipid — as in choline deficiency, whose reversal is the lipotropic action; or a failure of the secretory mechanism.
• Ethanol causes fatty liver by raising the NADH/NAD⁺ ratio, which inhibits fatty acid oxidation and the citric acid cycle and favours esterification. Prolonged steatosis may progress to fibrosis and cirrhosis.

The phospholipid route ties pathology back to structure: no phosphatidylcholine means no lipoprotein surface monolayer, so no VLDL can be assembled.

• All its carbon atoms are provided by acetyl-CoA. Synthesis is cytosolic and occurs in virtually all tissues — chiefly liver, intestine, adrenal cortex and reproductive tissues — with NADPH supplying reducing equivalents. About 700 mg is synthesised daily, more than the diet supplies.
• Five stages: acetyl-CoA → mevalonate (C6) → isoprenoid units (C5) → squalene (C30) → lanosterol → cholesterol (C27).
• The steroid nucleus cannot be degraded to CO₂ and water in the body. Cholesterol is eliminated only by conversion to bile acids or excretion as neutral steroids, about 1 g per day, roughly half by each route.

That negative fact — the ring cannot be broken down — is why reverse cholesterol transport, and therefore HDL, matters so much.

• Regulated at four levels: transcription, via SREBP, activated when cellular sterol is low and increasing transcription of both the reductase and the LDL receptor; regulated degradation of the enzyme protein; covalent modification — phosphorylation inactivates; and feedback inhibition by cholesterol, dietary or delivered in LDL.
• Insulin and thyroid hormone increase activity; glucagon and glucocorticoids decrease it.
• It is the target of the statins, competitive inhibitors which lower plasma LDL chiefly by an indirect route: falling intracellular cholesterol de-represses LDL-receptor synthesis, so the liver clears more LDL from blood.

Do not confuse with HMG-CoA SYNTHASE, the mitochondrial regulatory enzyme of ketogenesis, or with HMG-CoA lyase. Three enzymes, one substrate name, three different exam answers.

• The primary bile acids are cholic acid and chenodeoxycholic acid; 7α-hydroxylase is the rate-limiting enzyme, and it is feedback-inhibited by bile acids.
• Before secretion they are conjugated with glycine or taurine, which keeps them ionised and therefore better detergents at intestinal pH. Secondary bile acids — deoxycholic and lithocholic — are produced by intestinal bacteria.
• Enterohepatic circulation: 98–99% is reabsorbed in the ileum and returned to the liver, the pool cycling six to ten times a day, with only about 0.5 g lost daily in the feces.

Interrupting that cycle with bile acid sequestrants such as cholestyramine — or with soluble dietary fibre — forces the liver to convert more cholesterol into bile acids, and so lowers plasma cholesterol.

• Catalysed by aminotransferases (transaminases), all of which carry pyridoxal phosphate (PLP), a derivative of vitamin B₆, at the catalytic site, where it acts as the carrier of the amino group via an enzyme-bound Schiff base. The mechanism is “ping-pong” — alternate addition of a substrate and release of a product.
• The reaction is freely reversible, with an equilibrium constant close to unity, so it serves in both amino acid catabolism and biosynthesis. All the common amino acids except lysine, threonine, proline and hydroxyproline participate.
• Significance: since alanine is also a substrate for glutamate aminotransferase, the α-amino nitrogen of all amino acids that undergo transamination can be concentrated in glutamate — the only amino acid oxidatively deaminated at an appreciable rate. Transamination is therefore the collection system for nitrogen disposal.
• Clinically: AST (SGOT) is raised in myocardial infarction, ALT (SGPT) in viral hepatitis.

⭐ The single most reliably examined term in the whole subject — set in Section I of BOTH the 2019 and the 2020/21 paper. Say “reversible” and name pyridoxal phosphate in the first sentence.

• Synthesis of 1 mol of urea requires 3 mol of ATP, 1 mol each of ammonium ion and of aspartate, and employs five enzymes. NH₃, CO₂ and the amide nitrogen of aspartate provide the atoms of urea.
• 1 · CO₂ + NH₄⁺ + 2 ATP → carbamoyl phosphate (carbamoyl phosphate synthase I, the rate-limiting enzyme, active only with N-acetylglutamate) — mitochondrial
  2 · + ornithine → citrulline (ornithine transcarbamoylase) — mitochondrial
  3 · + aspartate + ATP → argininosuccinate (argininosuccinate synthase) — cytosolic
  4 · → arginine + fumarate (argininosuccinate lyase) — cytosolic
  5 · → urea + ornithine (arginase) — cytosolic
• Ornithine is regenerated, so there is no net loss or gain of ornithine, citrulline, argininosuccinate or arginine. The fumarate links the cycle to the citric acid cycle, and the aspartate-fumarate skeleton acts as a carrier of nitrogen rather than a consumable.
• Disorders of every enzyme are known, characterised by hyperammonemia, encephalopathy and respiratory alkalosis.

Ammonia intoxication is most severe when the block is at reactions 1 or 2, for if citrulline can be synthesized, some ammonia has already been removed by being covalently linked to an organic metabolite.

• Nitrogen equilibrium (balance = 0): protein synthesis = protein degradation — the normal adult state.
• Positive nitrogen balance: an excess of ingested over excreted nitrogen; synthesis exceeds degradation. Accompanies growth and pregnancy.
• Negative nitrogen balance: output exceeds intake. May follow surgery, advanced cancer, and the nutritional disorders kwashiorkor and marasmus.
• Related figures: humans turn over 1% to 2% of total body protein daily, principally muscle protein, and about 75% of the liberated amino acids are reutilized.

Excess amino acids are not stored — those not immediately incorporated into protein are rapidly degraded. That single fact is why nitrogen disposal must be continuous.

• It can use either NAD⁺ or NADP⁺. The reaction is reversible but strongly favours glutamate formation, and therefore serves in amino acid biosynthesis as well as catabolism.
• Allosterically inhibited by ATP, GTP and NADH; activated by ADP — it runs when the cell needs fuel, since its product α-ketoglutarate is a citric acid cycle intermediate.
• Its concerted action with glutamate aminotransferase is termed transdeamination, and it is the route by which the formation of ammonia from α-amino groups mainly occurs.

Although the equilibrium favours glutamate, the reaction is pulled in the deaminating direction because the ammonia is immediately consumed by carbamoyl phosphate synthase I. The urea cycle is what makes deamination possible.

• Normally only traces (10–20 μg/dL) are present in peripheral blood, because ammonia from enteric bacteria and from tissues is rapidly removed by the liver. Toxic levels arise if portal blood bypasses the liver — in severely impaired hepatic function, or through collateral portal-systemic links in cirrhosis — or in inherited urea cycle defects.
• Symptoms: tremor, slurred speech, blurred vision, coma and ultimately death.
• Mechanism: ammonia reacts with α-ketoglutarate to form glutamate; the resulting depletion of α-ketoglutarate impairs function of the TCA cycle in neurons.

This is the anaplerosis problem of the citric acid cycle in clinical dress — and it explains why hyperammonemia and hypoglycemia produce such similar neurological pictures: both are failures of neuronal ATP supply.

• Exclusively ketogenic: LEUCINE and LYSINE only.
  Both: isoleucine, phenylalanine, tryptophan, tyrosine.
  All the remainder are glucogenic.
• The reason for the division is a single irreversible reaction: pyruvate dehydrogenase. Carbon entering as acetyl-CoA cannot return to pyruvate, and the two carbons entering the citric acid cycle are balanced by two leaving as CO₂ — so there is no net oxaloacetate and therefore no net glucose.
• Entry points: pyruvate (Ala, Trp, Cys, Ser, Gly, Thr) · oxaloacetate (Asn, Asp) · α-ketoglutarate (Arg, His, Gln, Pro, Glu) · succinyl-CoA (Ile, Met, Val) · fumarate (Phe, Tyr) · acetyl-CoA/acetoacetyl-CoA (Leu, Lys, Phe, Trp, Tyr).

⚠️ TMU Lecture 19 slide 58 lists seven amino acids as “ketogenic”, including threonine, which Harper's Table 29-1 classifies as glycogenic. That is the “ketogenic or partly ketogenic” list; the lecture's own question slide asks for the exclusively ketogenic pair, which is Leu and Lys.

• Phenylalanine accumulates and undergoes transamination to phenylpyruvate, which accumulates in blood and tissues and is excreted in the urine; some is reduced to phenylacetate, which gives the characteristic odour to the urine.
• Brain damage: accumulation of phenylalanine in early life impairs normal development of the brain, partly because excess phenylalanine competes with other amino acids for transport across the blood-brain barrier.
• Treatment: mental retardation can be prevented by a rigid diet providing only enough phenylalanine for growth — a restriction, not an elimination, since phenylalanine is nutritionally essential.
• Types II and III are defects of dihydrobiopterin reductase, types IV and V of dihydrobiopterin biosynthesis; collectively the hyperphenylalaninemias.

⚠️ TMU Lecture 19 slides 59–60 call the enzyme “phenylalanine carboxylase”. That is an error — the lecture's own slide 66 and Harper's both give hydroxylase. Write hydroxylase in the exam.

• The overall determinant of the rate of de novo purine nucleotide biosynthesis is the concentration of PRPP, which depends on the availability of ribose 5-phosphate — from the pentose phosphate pathway — and on PRPP synthase, feedback inhibited by AMP, ADP, GMP and GDP.
• Its role differs between the two pathways: in purine synthesis it is the scaffold on which the ring is built; in pyrimidine synthesis it joins only after the ring is complete, at OMP.
• Because it is common to both, PRPP synthase is the pivot of the coordinated, mole-for-mole regulation of purine and pyrimidine synthesis.

Whenever PRPP rises, purine synthesis rises and urate follows. That one sentence explains both von Gierke disease (extra ribose 5-phosphate) and Lesch-Nyhan syndrome (PRPP not consumed by salvage).

• Mechanism 1 — phosphoribosylation by PRPP: Pu + PRPP → Pu-RP + PPi, catalysed by adenine phosphoribosyltransferase (adenine → AMP) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (hypoxanthine → IMP, guanine → GMP).
• Mechanism 2 — phosphoryl transfer from ATP: Pu-R + ATP → PuR-P + ADP, catalysed by adenosine kinase and deoxycytidine kinase.
• Liver is the major site of de novo synthesis and supplies purines to tissues incapable of it — brain, which has a low level of PRPP glutamyl amidotransferase, and erythrocytes and polymorphonuclear leukocytes, which cannot make 5-phosphoribosylamine at all.

Because the brain depends partly on salvage, a defect in HGPRT produces a neurological syndrome — Lesch-Nyhan. The tissue distribution of the enzyme predicts the phenotype.

• Why humans are susceptible: humans lack uricase, which in other mammals converts uric acid to the soluble product allantoin, so uric acid is the end product of purine catabolism in humans.
• Uric acid has a pKa of 5.8 — relatively insoluble as the free acid at acidic pH, more soluble as sodium urate near neutrality. This explains both uric acid stones in acid urine and deposition in cool peripheral joints.
• Causes: various genetic defects in PRPP synthase — elevated V_max, increased affinity for ribose 5-phosphate, or resistance to feedback inhibition — cause overproduction; but most cases of gout reflect abnormalities in RENAL handling of uric acid. Urate crystals are diagnostic.
• Treatment: allopurinol, which inhibits xanthine oxidase, so the more soluble hypoxanthine and xanthine are excreted instead.

Contrast pyrimidines: since the end products of pyrimidine catabolism are highly water-soluble, pyrimidine overproduction results in few clinical signs or symptoms. The difference is structural — a fused bicyclic ring versus a single small one.

• Mechanism of the overproduction: salvage normally consumes PRPP; when HGPRT fails, the accompanying rise in intracellular PRPP results in purine overproduction. The patient therefore both fails to recycle purines and makes too many new ones.
• The neurological features follow from the fact that brain has a low level of PRPP glutamyl amidotransferase and depends in part on exogenous purines.
• Causative mutations include deletions, frameshift mutations, base substitutions and aberrant mRNA splicing.

• Two mechanisms: retention hyperbilirubinemia, from overproduction (unconjugated), and regurgitation hyperbilirubinemia, from reflux into the bloodstream because of biliary obstruction (conjugated).
• Prehepatic — haemolytic anaemias; ↑ indirect, urine urobilinogen increased, urine bilirubin ABSENT (acholuric).
  Hepatic — hepatitis, cirrhosis; both fractions rise, ALT and AST markedly elevated.
  Posthepatic — gallstone in the common bile duct, cancer of the head of the pancreas; ↑ direct, urine bilirubin PRESENT (choluric), urobilinogen absent, pale stools, alkaline phosphatase elevated.
• Only unconjugated bilirubin crosses the blood-brain barrier — hence kernicterus; only conjugated bilirubin appears in urine.

Both rules follow from one property. Two solubilities, two diseases — reconstruct the whole laboratory table from that and you never need to memorise it.

• Liver: glucokinase traps large influxes of glucose from the hepatic portal vein — its K_m of 10 mM (against 0.1 mM for hexokinase) means it works only above the fasting level of 5 mM, and unlike hexokinase it is not inhibited by glucose 6-phosphate. High insulin/glucagon increases the activity of glycogen synthase and pyruvate dehydrogenase.
• Muscle: insulin increases glycogen synthase activity and protein synthesis. Except for short elevations in energy demand, the preferential fuel for muscle is fat.
• Adipose tissue: insulin induces lipoprotein lipase and inhibits hormone-sensitive lipase.
• Brain: burns glucose, as always.

The glucokinase kinetics are Unit 6 doing physiology: a high-K_m, non-inhibited enzyme takes up glucose only when there is a surplus, so the liver never competes with the brain.

• The first priority of metabolism is to provide sufficient glucose to the brain and red blood cells, and the blood glucose level must be maintained above 2.2 mM.
• Gluconeogenesis slows down; free fatty acids continue to be mobilized and the oxidation of fatty acids continues unabated, so oxaloacetate is drawn into gluconeogenesis and the surplus acetyl-CoA becomes ketone bodies. Muscle increases its reliance on free fatty acids and spares ketones for use by the brain.
• Fuel table (g/24 h), day 3 → day 40: brain glucose 100 → 40; brain ketone bodies 50 → 100; muscle-protein degradation 75 → 20; adipose lipolysis 180 → 180, unchanged; liver glucose output 150 → 80; liver ketone bodies 150 → 150.

Ketone bodies exist to spare protein. The brain switches fuel rather than reducing consumption, and the payoff is that muscle degradation falls from 75 to 20 g a day.

• Liver: hepatic output of glucose is increased because of stimulation of glycogenolysis and gluconeogenesis. The increased supply of FFA to the liver, combined with the removal of oxaloacetate by the gluconeogenic pathway, shunts excessive amounts of acetyl-CoA into the production of ketone bodies — at a rate much greater than is seen in starvation.
• Adipose tissue: the low ratio signals fasting, so lipolysis is unopposed; reduced lipoprotein lipase activity causes elevation of chylomicrons and VLDL; and too little glucose is taken up.
• Skeletal muscle: skeletal muscle needs insulin to keep proteins from being degraded; without it, protein synthesis is decreased and degradation is increased.

Starvation in the midst of plenty. Ketoacidosis exceeds starvation ketosis for two reasons: lipolysis is completely unopposed (insulin is the principal antilipolytic hormone), and the ketone bodies are not consumed, because no fuel switch occurs when blood glucose is high.

• Where they come from: chiefly the glycine cleavage complex of liver mitochondria, which splits glycine to CO₂ and NH₄⁺ and forms N⁵,N¹⁰-methylene tetrahydrofolate; the interconversion of serine and glycine by glycine hydroxymethyltransferase; the catabolism of histidine, whose N-formiminoglutamate (Figlu) transfers its formimino group to tetrahydrofolate; and the demethylation reactions of sarcosine.
• What they are used for: purine biosynthesis — N¹⁰-formyl-tetrahydrofolate supplies C2 and C8 of the purine ring, so antifolate drugs and glutamine analogs inhibit purine biosynthesis; thymidylate synthesis — the only reaction of pyrimidine nucleotide biosynthesis that requires a tetrahydrofolate derivative, in which the methylene group of N⁵,N¹⁰-methylene-tetrahydrofolate becomes the methyl group of TMP; and methylation reactions generally, via S-adenosylmethionine.
• Clinical significance: in folic acid deficiency, transfer of the formimino group is impaired and Figlu is excreted — excretion of Figlu following a dose of histidine can therefore be used to detect folic acid deficiency. Methotrexate blocks dihydrofolate reductase, so tetrahydrofolate cannot be regenerated after thymidylate synthase consumes it.

⭐ A proven 2019 Section I term. Because N⁵,N¹⁰-methylene-tetrahydrofolate is required for thymidylate synthesis, disorders of folate and vitamin B₁₂ metabolism result in deficiencies of TMP — and hence in megaloblastic anaemia: cells that grow but cannot divide.

• Structure: the octamer is two each of H2A, H2B, H3 and H4. In the nucleosome, the DNA is supercoiled in a LEFT-HANDED helix over the surface of the disk-shaped histone octamer; 1.75 superhelical turns of DNA are wrapped around the surface, protecting 145 to 150 bp and forming the nucleosome core particle.
• Assembly: the (H3-H4)₂ tetramer itself can confer nucleosome-like properties on DNA; addition of two H2A-H2B dimers stabilizes the primary particle. H1 is NOT part of the core — it is the histone least tightly bound to chromatin, easily removed with a salt solution, binds the linker DNA, and is not necessary for reconstitution of the nucleosome core.
• Function: condensation. The human haploid genome consists of about 3 × 10⁹ bp and about 1.7 × 10⁷ nucleosomes, and must be compressed about 8000-fold to generate the structure of a condensed metaphase chromosome.

Packaging IS regulation. Acetylation of lysines in the histone tails reduces their positive charge, decreases affinity for DNA and disrupts nucleosomal structure, giving transcription factors access — see [[unit 26]].

• Why they are needed — the end-replication problem: DNA polymerases synthesise only 5′→3′ and cannot initiate a chain, so on the lagging strand the final RNA primer at the chromosome end is removed and the gap cannot be filled. Every round of replication would otherwise shorten the chromosome; telomeres are expendable non-coding repeats that absorb the loss.
• Telomerase — a multisubunit RNA template-containing complex related to viral RNA-dependent DNA polymerases (reverse transcriptases) — is the enzyme responsible for telomere synthesis and thus for maintaining the length of the telomere.
• Since telomere shortening has been associated with both malignant transformation and aging, this enzyme has become an attractive target for cancer chemotherapy and drug development.

Do not confuse with the centromere — an A-T-rich region of repeated DNA bound by CENP-A nucleotomes to form the kinetochore, the anchor for the mitotic spindle.

• Denaturation is accompanied by HYPERCHROMICITY OF DENATURATION — an increase in the optical absorbance of the purine and pyrimidine bases, because the bases unstack — and by a loss of viscosity, since double-stranded DNA exhibits properties of a rigid rod.
• DNA rich in G-C pairs, which have three hydrogen bonds, melts at a higher temperature than that rich in A-T pairs, which have two.
• A 10-fold increase of monovalent cation concentration increases the T_m by 16.6 °C, by neutralizing the intrinsic interchain repulsion between the highly negatively charged phosphates. Formamide destabilizes hydrogen bonding between bases, thereby lowering the T_m.
• Separated strands renature when appropriate temperature and salt conditions are achieved; this reannealing is often referred to as HYBRIDIZATION — the basis of Southern (DNA/DNA) and Northern (RNA/DNA) blotting.

Note the dimensions of B-DNA that go with it: 10 bp per turn, 3.4 nm (34 Å) per turn, 2 nm (20 Å) wide, found under physiologic conditions of low salt and high hydration.

• It follows directly from base pairing: since A pairs only with T and G only with C, each strand already contains the information needed to rebuild the other.
• Replication starts at an origin (ori) — bound by dnaA in E. coli, by the origin recognition complex (ORC) in eukaryotes — beside an ~80-bp A+T-rich DNA unwinding element (DUE), and proceeds bidirectionally as a replication bubble.
• Because DNA polymerases synthesise only 5′→3′ and the strands are antiparallel, the polymerase functions asymmetrically: the leading strand is synthesized continuously and the lagging strand in short 1–5 kb Okazaki fragments, each primed by RNA and later sealed by DNA ligase.

Two constraints on DNA polymerase — 5′→3′ only and cannot initiate a chain — generate every complication at the fork, and, at a chromosome end, the end-replication problem that [[telomeres]] exist to solve.

• DNA-dependent RNA polymerase attaches at this specific site on the template strand. This is followed by initiation of RNA synthesis at the starting point, and the process continues until a termination sequence is reached.
• The promoter determines two things: where transcription is to commence along the DNA, and how frequently this event is to occur.
• In bacteria, promoters are approximately 40 nucleotides long, with an eight-nucleotide-pair sequence about 35 bp upstream of the start site and a six-nucleotide-pair A+T-rich sequence about 10 nucleotides upstream. The σ subunit helps the core enzyme recognize and bind to the promoter region.
• In eukaryotes, promoters are more complex: TATA box, initiator sequence (Inr) and downstream promoter element (DPE), plus promoter-proximal elements (50–200 bp) and distal enhancers and repressors (1000–10⁵ bp). The TATA box has a particularly rigid requirement for both position and orientation, and is bound by TFIID through its TATA-binding protein (TBP) subunit.

⭐ Set in Section I of BOTH papers. Distinguish it from the transcription unit — the region of DNA that includes the signals for transcription initiation, elongation and termination — which is often set alongside it.

• Catalysed by DNA-dependent RNA polymerase, the enzyme responsible for the polymerization of ribonucleotides into a sequence complementary to the template strand of the gene, in three general steps: initiation, elongation and termination.
• Key features: 5′→3′ polarity, with the template strand read 3′→5′; substrates ATP, GTP, CTP and UTP, U replacing T; and a primer is NOT involved, as RNA polymerases can initiate synthesis de novo. Only the template strand is copied, and it will not necessarily be the same strand of the double helix for every gene.
• The transcription bubble is 20 bp and the whole complex covers 30–75 bp. Bacterial core enzyme α₂ββ′; holoenzyme α₂ββ′σ.
• Termination in bacteria is by an intrinsic terminator — an inverted, hyphenated repeat followed by a stretch of AT base pairs, whose transcript forms a hairpin that causes RNA polymerase to pause — or by rho, an ATP-dependent RNA-stimulated helicase.

Mammalian cells possess three distinct nuclear DNA-dependent RNA polymerases: Pol I (rRNA), Pol II (mRNA), Pol III (tRNA, 5S). α-Amanitin distinguishes them.

• The intron RNA sequences are cleaved out of the transcript, and the exons are appropriately spliced together IN THE NUCLEUS before the resulting mRNA molecule appears in the cytoplasm for translation.
• Splicing depends on consensus sequences at the splice junctions and an internal branch site, and is carried out by the spliceosome, assembled from snRNAs (small nuclear RNAs) and snRNPs (small nuclear ribonucleoprotein particles). The catalysis is performed by RNA — the snRNAs are ribozymes.
• Alternative splicing provides for different mRNAs, so one gene may specify several proteins.

The related device of alternative promoter utilization: in the glucokinase gene, the β-cell promoter and exon 1B lie about 30 kbp upstream of the liver promoter and exon 1L, while exons 2–10 are identical and the proteins have identical kinetic properties — two jobs, two control systems, one coding sequence.

• 1 · Capping. Mammalian mRNA molecules contain a 7-methylguanosine cap structure at their 5′ terminal. The 5′ cap is required both for efficient translation initiation and protection of the 5′ end from attack by 5′→3′ exonucleases.
• 2 · Polyadenylation. The mRNA is first cleaved about 20 nucleotides downstream from an AAUAAA sequence; poly(A) polymerase adds a tail subsequently extended to about 200 A residues. The poly(A) tail both protects the 3′ end from 3′→5′ exonuclease attack and facilitates translation. (Histone mRNA lacks a poly(A) tail.)
• 3 · Splicing — introns removed, exons joined, by the spliceosome.
• The cap and poly(A) tail structures have a synergistic effect on protein synthesis, because initiation factors bridge them and circularise the message.

Each end is protected against the exonuclease that attacks from its own direction, and both promote translation. Circularisation is also a quality check — only a completed, properly processed transcript can be circularised.

• Of the 64 possible codons (4³), three are nonsense codons used as termination signals — UAA, UAG, UGA, and the remaining 61 code for the 20 naturally occurring amino acids. Since 61 > 20, most amino acids have several codons: six different codons — UCU, UCC, UCA, UCG, AGU, AGC — all specify serine, while methionine and tryptophan have a single codon.
• Degeneracy is NOT ambiguity. For any specific codon, only a single amino acid is specified; with rare exceptions the genetic code is unambiguous. The distinction between ambiguity and degeneracy is an important concept. The code is degenerate but unambiguous — translation is reliable, back-translation is not.
• In general, the third nucleotide in a codon is less important than the first two in determining the specific amino acid to be incorporated — the basis of WOBBLE.

The redundancy is a shock absorber — many base changes are silent — and it allows 31 cytoplasmic tRNA species to read 61 codons (mitochondria manage with 22).

• The anticodon region (arm) consists of seven nucleotides, and it recognizes the three-letter codon in mRNA. Reading 3′→5′, the loop is variable base (N) — modified purine (Pu*) — XYZ (the anticodon) — pyrimidine (Py) — pyrimidine (Py).
• This direction of reading the anticodon is 3′→5′, whereas the genetic code is read 5′→3′, since the codon and the anticodon loop are ANTIPARALLEL in their complementarity, just like all other intermolecular interactions between nucleic acid strands.
• Significance: for a given codon in the mRNA, only a single species of tRNA molecule possesses the proper anticodon; since each tRNA can be charged with only one specific amino acid, each codon therefore specifies only one amino acid — this is why the code is unambiguous. However, some tRNA molecules can utilize the anticodon to recognize more than one codon — wobble.
• The other arms: acceptor arm (3′-CCA, carries the amino acid), TψC arm (binds the ribosomal surface), D arm (recognised by the aminoacyl-tRNA synthetase).

• The degeneracy of the genetic code resides mostly in this last nucleotide. For example, the two codons for arginine, AGA and AGG, can bind to the same anticodon. Inosine, one of the unusual bases of tRNA, is the classic wobble base.
• Consequence: one tRNA can read several codons, so the cytoplasmic translation system possesses a full complement of 31 tRNA species for 61 codons, and mitochondria require only 22 tRNA molecules.

Degeneracy and wobble are the same molecular property described twice — the code is degenerate BECAUSE the third pairing is loose. It is also why third-base mutations are so often silent.

• It forms an activated intermediate of aminoacyl-AMP-enzyme complex, and the amino acid remains attached to its specific tRNA in an ESTER linkage at the 3′-CCA of the acceptor arm. Two high-energy phosphate bonds are spent.
• The charging reactions have an error rate of less than 10⁻⁴ and so are extremely accurate.

That accuracy is critical because the ribosome inspects only the codon-anticodon pairing, never the identity of the attached amino acid. The fidelity of the genetic code is enforced at charging, not at the ribosome.

• It yields POLYCISTRONIC mRNA — transcribed into one large mRNA molecule containing multiple independent translation start (AUG) and stop (UAA) codons for each cistron — giving coordinate expression. A cistron is the genetic unit encoding one polypeptide: the “one cistron, one subunit” concept, refining “one gene, one enzyme”.
• The lac operon is the model: lacZ (β-galactosidase), lacY (a permease, for the permeation of lactose into the cell) and lacA (a thiogalactoside transacetylase), with lacI, encoding the repressor, outside the operon.

Operons do not occur in eukaryotes, because cap-dependent scanning permits only one translation initiation site per mRNA. One feature of initiation determines an entire architecture of gene organisation.

• Structural genes: lacZ — β-galactosidase; lacY — a permease, the permeation of lactose into the cell; lacA — a thiogalactoside transacetylase, transcribed as one polycistronic mRNA.
• Negative control: lacI encodes the lac operon repressor protein — four identical subunits of molecular weight 38,000, with a high affinity for the operator locus. The operator locus is a region of double-stranded DNA 27 base pairs long, with a twofold rotational symmetry and an inverted palindrome; LacI repressor binding is 17 base pairs, and only two subunits of the repressor appear to bind. Addition of lactose or of a gratuitous inducer such as IPTG causes derepression.
• Positive control: catabolite gene activator protein (CAP), also referred to as the cAMP regulatory protein (CRP), in conjunction with cAMP.
• Kinetics: lac-specific mRNAs are fully induced within 5–6 minutes; β-galactosidase protein is maximal within 10 minutes — a type A response.

Maximal activity requires lactose present AND glucose absent — an AND gate built from one regulator that acts positively (the cAMP-CRP complex) and one that acts negatively (the LacI repressor).

• When bacteria are exposed to both lactose and glucose as sources of carbon, they first metabolize the glucose and then temporarily stop growing, until the genes of the lac operon become induced and they gain the ability to metabolize lactose as a usable energy source.
• It is mediated by a catabolite gene activator protein (CAP) in conjunction with cAMP. When glucose or glycerol is present in concentrations sufficient for growth, the bacteria will lack sufficient cAMP to bind to CAP, because the glucose inhibits adenylyl cyclase, the enzyme that converts ATP to cAMP.
• The CAP-cAMP regulator is acting as a POSITIVE regulator.

cAMP means the same thing here as in the hepatocyte of [[unit 12]]: glucose is scarce. The same messenger with the same meaning, three billion years apart.

• Derepression is removal of a repressor, and works because a double negative has the effect of acting as a positive.
• Inducible genes have relatively low basal rates of transcription; housekeeping genes are expressed constantly; a constitutive mutation makes an inducible gene expressed permanently.
• cis-acting elements are DNA sequences acting only on the molecule they are part of; trans-acting factors are diffusible proteins acting on any copy. This is why a lacI mutation can be complemented and an operator mutation cannot.

A gene under negative control is off by default and needs no continuous input to stay off, and can be switched on by the substrate itself — which is exactly what lactose does.

• Histone acetylation and deacetylation occur on lysine residues in the amino terminal tails of histone molecules. Acetylation reduces the positive charge of these tails, decreases the binding affinity of histone for the negatively charged DNA, and causes disruption of nucleosomal structure, allowing access of transcription factors to cognate regulatory DNA elements and enhancing binding of the basal transcription machinery to the promoter. Histone deacetylation would have the opposite effect.
• Methylation of deoxycytidine residues in the sequence 5′-mCpG-3′ silences genes: in mouse liver, only the unmethylated ribosomal genes can be expressed, and many animal viruses are not transcribed when their DNA is methylated.

Because both marks are copied through cell division, this level of control is heritable — which is what a repressor protein can never be, since it must be continuously present and is diluted at every division.

• General principles: binding must be of high affinity to the specific site and of low affinity to other DNA; binding is usually by a dimer, matching the twofold symmetry of a palindromic site; and the protein-DNA interactions are maintained by hydrogen bonds and van der Waals forces.
• Helix-turn-helix — illustrated by Cro: the monomer consists of three antiparallel β sheets (β1–β3) and three α-helices (α1–α3); the α3 and α2 helices are held at about 90 degrees to each other by a turn of four amino acids; the α3 helix of Cro is the DNA recognition surface. The distance between comparable points on the two DNA α-helices is 34 Å — one turn of B-DNA.
• Zinc finger — a domain folded around a zinc ion stabilising a short recognition helix.
• Leucine zipper — a helical wheel consisting of seven spokes that correspond to the seven amino acids, with leucine residues at every seventh position; it mediates dimerisation while an adjacent basic region binds DNA.

All three converge on the same solution — presenting a short α-helix to the major groove — because the edges of the base pairs are exposed there and the groove is about the width of an α-helix.