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Mecanismos de Bioquímica · Metabolic pathways

Glicólise e ciclo do ácido cítrico

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O que você vai aprender

  • Draw the phosphoryl transfers of hexokinase, phosphoglycerate kinase, succinyl-CoA synthetase and pyruvate kinase as in-line attacks on phosphorus.
  • Draw the enediol isomerisations, the aldolase Schiff-base retro-aldol and the enolase and fumarase eliminations and additions with the acid and base the active site supplies.
  • Explain how glyceraldehyde-3-phosphate dehydrogenase and isocitrate dehydrogenase couple hydride transfer to NAD⁺ with thioester formation or decarboxylation.
  • Draw the citrate synthase condensation and the aconitase dehydration–rehydration, and explain why each step's electrophile, nucleophile and stereochemistry are what they are.
  • Calculate ATP yields, ΔG from ΔG°′ and concentrations, and ΔG°′ from reduction potentials for glycolysis and the citric acid cycle.

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Glycolysis: invest two ATP, then split

Glycolysis turns one glucose into two pyruvate. In the first half the cell spends two ATP. Hexokinase phosphorylates the 6-OH of glucose: the oxygen, deprotonated by an aspartate, attacks the terminal (γ) phosphorus of Mg²⁺-ATP in line with the bond to ADP, which leaves. The phosphate traps glucose in the cell as an anion and starts its activation. Phosphoglucose isomerase converts the aldose glucose 6-phosphate into the ketose fructose 6-phosphate, phosphofructokinase-1 adds a second phosphate at C1, and aldolase cuts fructose 1,6-bisphosphate (shown) between C3 and C4 into two three-carbon phosphates: dihydroxyacetone phosphate (C1–C3) and glyceraldehyde 3-phosphate (C4–C6). Hexokinase closes around glucose before it transfers phosphate; that induced fit keeps water out, so ATP is not simply hydrolysed.

Enediols and Schiff bases: carbonyl chemistry in an active site

Enzymes use the same carbonyl chemistry as the flask, with a carboxylate as the base and a protonated histidine or lysine as the acid. An isomerase removes the proton α to a carbonyl while an acid protonates the carbonyl oxygen, giving a cis-enediol (the triose phosphate isomerase intermediate is shown). Returning a proton to the other carbon moves the carbonyl one carbon along: aldose becomes ketose, or dihydroxyacetone phosphate becomes glyceraldehyde 3-phosphate. The enediol can also expel the neighbouring phosphate to give toxic methylglyoxal, which triose phosphate isomerase prevents by closing a loop over the intermediate. Class I aldolase first condenses a lysine with the C2 ketone of fructose 1,6-bisphosphate to give a protonated Schiff base (an iminium ion). The iminium is a far better electron sink than a ketone, so when a base removes the C4 hydroxyl proton the C3–C4 bond breaks, releasing glyceraldehyde 3-phosphate and leaving an enamine that is protonated on carbon. Sodium borohydride reduces this iminium to a stable amine and kills the enzyme, the classic evidence for the Schiff base.

Oxidation through a thioester

Glyceraldehyde-3-phosphate dehydrogenase cannot take a hydride from the aldehyde directly, because nothing would push it off. A cysteine thiolate first adds to the aldehyde to give a thiohemiacetal; its oxygen lone pair then re-forms C=O and pushes the C–H hydride to C4 of the nicotinamide ring of NAD⁺, giving NADH and a thioester. The oxidation energy is kept in that thioester instead of being lost to a free carboxylic acid. Inorganic phosphate then attacks the thioester carbonyl, and the tetrahedral intermediate expels the cysteine to give the acyl phosphate 1,3-bisphosphoglycerate (shown). Arsenate can replace phosphate here, but the acyl arsenate hydrolyses at once, so the following ATP-forming step is bypassed.

Phosphoryl-transfer potential and the payoff phase

A phosphate can be passed to ADP only by a donor whose hydrolysis is more exergonic than that of ATP (ΔG°′ ≈ −30.5 kJ/mol). The acyl phosphate of 1,3-bisphosphoglycerate (≈ −49.3 kJ/mol) gives its phosphate to ADP on phosphoglycerate kinase, leaving the carboxylate of 3-phosphoglycerate. Phosphoglycerate mutase moves the phosphate to C2 through 2,3-bisphosphoglycerate, using a phosphohistidine. Enolase removes the C2 proton with a lysine, forming an aci-carboxylate (enolate) held by Mg²⁺, and a glutamate protonates the C3 hydroxyl as it leaves: an E1cB dehydration that gives phosphoenolpyruvate (shown). PEP (≈ −61.9 kJ/mol) is the strongest donor because the product it leaves, enolpyruvate, tautomerises to the far more stable keto form; pyruvate kinase transfers the phosphate to ADP and then protonates the enolate on carbon. Per glucose: −2 ATP (hexokinase, phosphofructokinase-1), +2 (phosphoglycerate kinase) and +2 (pyruvate kinase), so 2 ATP net and 2 NADH.

The citric acid cycle: condense, isomerise, decarboxylate

Citrate synthase joins acetyl-CoA to oxaloacetate. An aspartate removes a proton from the acetyl methyl group while a histidine supports the oxygen, giving the enol; the enol carbon adds to the ketone carbonyl of oxaloacetate, which a second histidine protonates. That is an aldol-type addition onto a ketone, not a Claisen attack on the thioester. Oxaloacetate binds first and creates the acetyl-CoA site, so acetyl-CoA is not hydrolysed wastefully; hydrolysis of the citryl-CoA thioester then makes the reaction strongly exergonic. Citrate (shown) is achiral, but its two CH₂CO₂⁻ arms are enantiotopic, and aconitase works only on the arm that came from oxaloacetate. Aconitase dehydrates citrate by E1cB (a serine alkoxide removes the CH₂ proton, a histidine protonates the leaving hydroxyl) to cis-aconitate, which flips by 180° in the active site; the same residues then add water the other way round, giving (2R,3S)-isocitrate. Isocitrate dehydrogenase oxidises the secondary alcohol to a ketone with NAD⁺, which turns the middle carboxylate into a β-keto acid that loses CO₂; a tyrosine protonates the enolate to give α-ketoglutarate.

Regenerating oxaloacetate and counting the energy

α-Ketoglutarate dehydrogenase releases the second CO₂ and makes succinyl-CoA. Succinyl-CoA synthetase lets phosphate attack the thioester to give succinyl phosphate, passes the phosphoryl group to a histidine and then to GDP (or ADP): the cycle's one substrate-level phosphorylation. Succinate dehydrogenase uses a covalently bound FAD, because the fumarate/succinate couple (E°′ ≈ +0.031 V) is far too positive to reduce NAD⁺ (E°′ ≈ −0.320 V). Fumarase adds water anti across fumarate through an aci-carboxylate (carbanion) intermediate, giving only L-(S)-malate. Malate dehydrogenase oxidises malate to oxaloacetate with NAD⁺ although ΔG°′ ≈ +29.7 kJ/mol; citrate synthase keeps oxaloacetate so scarce that ΔG in the cell is near zero. Use ΔG = ΔG°′ + RT ln Q and ΔG°′ = −nFΔE°′ (F = 96.485 kJ mol⁻¹ V⁻¹) for the numbers. With about 2.5 ATP per NADH and 1.5 per FADH₂, one acetyl-CoA gives 3 × 2.5 + 1.5 + 1 = 10 ATP equivalents.

What to carry forward

Every step of glycolysis and the citric acid cycle is ordinary carbonyl, enol and phosphoryl chemistry: find the electron sink (C=O, iminium, thioester, carboxylate), identify which residue removes or donates each proton, and track where phosphate, hydride and CO₂ go. Then the energy bookkeeping follows from ΔG°′, concentrations and reduction potentials.

Problemas de prática (40)

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Desenhe o mecanismo 18

Citric acid cycle: carbon chemistry

  • Aconitase: E1cB dehydration of citrate to cis-aconitateDraw the serine-mediated deprotonation of citrate to an aci-carboxylate and the histidine-assisted loss of the C3 hydroxyl that gives cis-aconitate.
  • Aconitase: rehydration of cis-aconitate to isocitrateDraw the conjugate addition of water to cis-aconitate and the protonation of the aci-carboxylate that gives (2R,3S)-isocitrate.
  • Citrate synthase: enolisation of acetyl-CoA and addition to oxaloacetateDraw the enolisation of acetyl-CoA by the aspartate–histidine pair and the addition of the enol carbon to the ketone of oxaloacetate that gives citryl-CoA.
  • Citrate synthase: hydrolysis of citryl-CoA to citrateDraw the base-assisted addition of water to the citryl-CoA thioester, the loss of the CoA thiolate and the proton transfer that gives citrate and coenzyme A.
  • Isocitrate dehydrogenase: oxidation, decarboxylation and enolate protonationDraw the hydride transfer from isocitrate to NAD⁺, the decarboxylation of the β-keto acid oxalosuccinate and the protonation of the enolate that gives α-ketoglutarate.

Glycolysis: investment and cleavage

  • Class I aldolase: retro-aldol cleavage of the fructose 1,6-bisphosphate Schiff baseDraw the base-triggered C3–C4 cleavage of the aldolase iminium that releases glyceraldehyde 3-phosphate and the enamine, then the C-protonation that gives the dihydroxyacetone phosphate iminium.
  • Class I aldolase: Schiff base formation with fructose 1,6-bisphosphateDraw the addition of the lysine amine to the C2 ketone of fructose 1,6-bisphosphate, the proton transfers to the carbinolamine and the loss of water that gives the protonated Schiff base.
  • Hexokinase: phosphoryl transfer from ATP to glucoseDraw the in-line attack of the glucose 6-hydroxyl on the γ-phosphorus of ATP, deprotonated by an aspartate, that gives glucose 6-phosphate and ADP.
  • Phosphoglucose isomerase: glucose 6-phosphate to fructose 6-phosphate through an enediolDraw the base-catalysed formation of the cis-enediol from open-chain glucose 6-phosphate and its collapse to the ketose fructose 6-phosphate, with the glutamate moving the C2 proton to C1.
  • Triose phosphate isomerase: dihydroxyacetone phosphate to glyceraldehyde 3-phosphateDraw the enediol mechanism by which triose phosphate isomerase converts dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate.

Glycolysis: payoff phase

  • Enolase: E1cB dehydration of 2-phosphoglycerate to phosphoenolpyruvateDraw the lysine-mediated deprotonation of 2-phosphoglycerate to its aci-carboxylate and the glutamate-assisted loss of the C3 hydroxyl that gives phosphoenolpyruvate.
  • Glyceraldehyde-3-phosphate dehydrogenase: phosphorolysis of the thioesterDraw the addition of phosphate to the acyl-enzyme thioester and the collapse of the tetrahedral intermediate that releases 1,3-bisphosphoglycerate and the cysteine thiolate.
  • Glyceraldehyde-3-phosphate dehydrogenase: thiohemiacetal formation and hydride transfer to NAD⁺Draw the cysteine addition to glyceraldehyde 3-phosphate and the hydride transfer from the thiohemiacetal to NAD⁺ that gives the acyl-enzyme thioester and NADH.
  • Phosphoglycerate kinase: phosphoryl transfer from 1,3-bisphosphoglycerate to ADPDraw the attack of ADP on the acyl-phosphate phosphorus of 1,3-bisphosphoglycerate that makes ATP and 3-phosphoglycerate.
  • Pyruvate kinase: phosphoryl transfer from PEP to ADP and ketonisationDraw the phosphoryl transfer from phosphoenolpyruvate to ADP and the C-protonation of the pyruvate enolate that gives pyruvate.

Citric acid cycle: energy capture

  • Fumarase: hydration of fumarate to L-malateDraw the base-assisted addition of water to fumarate through an aci-carboxylate intermediate and its protonation to L-malate.
  • Malate dehydrogenase: oxidation of L-malate to oxaloacetateDraw the concerted removal of the hydroxyl proton by histidine and hydride transfer from L-malate to NAD⁺ that gives oxaloacetate.
  • Succinyl-CoA synthetase: thioester to acyl phosphate to phosphohistidine to GTPDraw how the energy of the succinyl-CoA thioester is passed through succinyl phosphate and a phosphohistidine to make GTP.

Preveja o produto 6

Citric acid cycle: carbon chemistry

  • What does aconitase release?Predict the product aconitase forms when it rehydrates cis-aconitate.
  • Put the cycle intermediates in orderOrder six citric acid cycle intermediates by the sequence in which they are formed, starting from citrate.

Glycolysis: investment and cleavage

  • What does aldolase make?Predict the two triose phosphates formed when aldolase cleaves fructose 1,6-bisphosphate between C3 and C4.

Glycolysis: payoff phase

  • What does enolase make?Predict the product of the enolase dehydration of 2-phosphoglycerate.
  • The phosphoglycerate mutase intermediatePredict the bisphosphate intermediate of the phosphohistidine-dependent phosphoglycerate mutase.

Citric acid cycle: energy capture

  • Which malate does fumarase make?Predict the configuration of the malate formed by fumarase hydration of achiral fumarate.

Classificar 1

  • The cycle's substrate-level phosphorylationClassify which citric acid cycle enzyme makes a nucleoside triphosphate by substrate-level phosphorylation.

Comparar 2

Glycolysis: payoff phase

  • Rank phosphoryl-transfer potentialRank glycolytic phosphate compounds by phosphoryl-transfer potential and decide which can phosphorylate ADP.

Citric acid cycle: energy capture

  • Why FAD, not NAD⁺, for succinate?Compare reduction potentials to explain why succinate dehydrogenase uses FAD rather than NAD⁺.

Calcular 5

Glycolysis: investment and cleavage

  • Is the aldolase step downhill in the cell?Calculate ΔG for the aldolase reaction from ΔG°′ and concentrations, including the effect of one substrate giving two products.

Glycolysis: payoff phase

  • Net ATP from glycolysis with arsenateCalculate the net ATP yield of glycolysis when arsenate replaces phosphate in the glyceraldehyde-3-phosphate dehydrogenase step.
  • Net ATP from glycolysisCount the ATP spent and made in glycolysis to obtain the net yield per glucose.

Citric acid cycle: energy capture

  • ATP equivalents per acetyl-CoACalculate the ATP equivalents produced by one turn of the citric acid cycle from its reduced carriers and GTP.
  • ΔG°′ for oxidising succinate with NAD⁺Calculate ΔG°′ for a hypothetical NAD⁺-linked oxidation of succinate from standard reduction potentials.

Encontre o erro 2

Citric acid cycle: carbon chemistry

  • Find the error in a citrate synthase mechanismDiagnose a citrate synthase proposal in which the nucleophile and electrophile are reversed.

Glycolysis: payoff phase

  • Find the error in a GAPDH mechanismDiagnose why a hydride cannot be removed from glyceraldehyde 3-phosphate before the cysteine adds.

Interprete as evidências 2

Glycolysis: investment and cleavage

  • What does the borohydride experiment show?Interpret the sodium borohydride trapping experiment as evidence for a lysine Schiff base in class I aldolase.

Citric acid cycle: carbon chemistry

  • Where does the C4 label of glucose go?Trace a labelled glucose carbon through glycolysis and pyruvate dehydrogenase to the step that releases it as CO₂.

Trabalhe de trás para frente 1

  • Which substrate gives DHAP and glyceraldehyde?Work back from the products of an aldolase C3–C4 cleavage to the phosphorylated ketohexose that was cleaved.

Explique por quê 3

Citric acid cycle: carbon chemistry

  • How can aconitase tell citrate's arms apart?Explain how an enzyme distinguishes the two enantiotopic arms of achiral citrate.

Glycolysis: investment and cleavage

  • Why phosphorylate glucose first?Explain why glycolysis begins by spending ATP to phosphorylate glucose at C6.
  • Why xylose makes hexokinase hydrolyse ATPUse the xylose experiment to explain how induced fit protects ATP from hydrolysis in hexokinase.

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