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Mecanismos de Bioquímica · Metabolic pathways

Metabolismo de lipídios e nucleotídeos

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O que você vai aprender

  • Draw the four reactions of a β-oxidation round (flavin dehydrogenation, hydration, NAD⁺ oxidation and thiolysis) with the acid, base and cofactor each active site supplies.
  • Draw the decarboxylative Claisen condensation, the two reductions and the dehydration of fatty acid synthesis, and contrast them with β-oxidation in carrier, cofactor, stereochemistry and compartment.
  • Explain how HMG-CoA links acetyl-CoA to ketone bodies and to cholesterol, and why the liver exports ketone bodies.
  • Draw phosphoryl and pyrophosphoryl transfers from ATP (adenylate kinase, nucleoside diphosphate kinase, PRPP synthetase, ATP hydrolysis) as in-line attacks on a chosen phosphorus.
  • Calculate ATP yields, NADPH costs, ΔG of ATP hydrolysis in the cell and the adenylate energy charge.
  • Explain the radical chemistry of ribonucleotide reductase and the one-carbon chemistry of thymidylate synthase, and how hydroxyurea and 5-fluoro-dUMP stop them.

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Activating a fatty acid and moving it into the mitochondrion

A free fatty acid is first made into a thioester. Acyl-CoA synthetase lets the carboxylate attack the α-phosphorus of ATP, giving an acyl adenylate and pyrophosphate; coenzyme A then displaces AMP. Pyrophosphatase hydrolyses the pyrophosphate, so activation costs two phosphoanhydride bonds (two ATP equivalents). The inner mitochondrial membrane does not let acyl-CoA through. Carnitine palmitoyltransferase I, on the outer membrane, moves the acyl group from CoA to the hydroxyl of carnitine (palmitoylcarnitine is shown); a translocase carries the acylcarnitine across the inner membrane, and carnitine palmitoyltransferase II returns the acyl group to a mitochondrial CoA. Malonyl-CoA, the first intermediate of fatty acid synthesis, inhibits carnitine palmitoyltransferase I, so a cell that is making fatty acids does not burn them at the same time.

One round of β-oxidation

Each round removes two carbons as acetyl-CoA and runs through four reactions; the drawing shows, from left to right, butyryl-CoA and the three intermediates of its β-oxidation. Acyl-CoA dehydrogenase uses a glutamate to remove an α-proton, which the thioester makes acidic, while the β-hydrogen moves as a hydride to N5 of FAD: the product is the trans-2-enoyl-CoA. Enoyl-CoA hydratase adds water across the C=C bond: a glutamate-activated water attacks the β-carbon in a conjugate addition, and the thioester enolate is protonated at the α-carbon, giving the L-(S)-3-hydroxyacyl-CoA. 3-Hydroxyacyl-CoA dehydrogenase then oxidises the alcohol to a ketone with NAD⁺, a histidine taking the O–H proton while the C–H hydrogen moves as hydride to C4 of the nicotinamide. Thiolase finishes the round: an active-site cysteine adds to the ketone carbonyl, the tetrahedral intermediate expels the enolate of acetyl-CoA (a retro-Claisen cleavage) and a second cysteine protonates it, leaving the shortened acyl group on the enzyme; coenzyme A then takes it back as the acyl-CoA.

Counting the yield, odd chains and double bonds

Palmitoyl-CoA (C16) needs seven rounds and gives 8 acetyl-CoA, 7 NADH and 7 FADH₂ (the flavin electrons reach ubiquinone through electron-transferring flavoprotein). With about 2.5 ATP per NADH, 1.5 per FADH₂ and 10 per acetyl-CoA oxidised in the citric acid cycle, that is 80 + 17.5 + 10.5 = 108 ATP, or 106 net after the two ATP equivalents of activation. An odd-chain acyl-CoA ends as propionyl-CoA, which a biotin-dependent carboxylase converts into (S)-methylmalonyl-CoA; an epimerase gives the (R) isomer, and the vitamin B₁₂ (adenosylcobalamin) radical enzyme methylmalonyl-CoA mutase rearranges it to succinyl-CoA. A natural cis double bond at an odd position, as in oleate, ends up as a cis-Δ³-enoyl-CoA that acyl-CoA dehydrogenase cannot use; enoyl-CoA isomerase moves it to the trans-Δ² position so the round can continue, skipping one FAD reduction.

HMG-CoA: ketone bodies and the route to cholesterol

Thiolase run in the condensing direction joins two acetyl-CoA into acetoacetyl-CoA. HMG-CoA synthase first transfers an acetyl group to an active-site cysteine; a glutamate removes a methyl proton from that acetyl-enzyme, and the enolate adds to the ketone carbonyl of acetoacetyl-CoA. A histidine protonates the new alkoxide, and hydrolysis of the cysteine thioester releases (S)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA, shown as its N-acetylcysteamine model). In liver mitochondria HMG-CoA lyase runs a retro-aldol: the C3 alkoxide re-forms a ketone and expels the enolate of acetyl-CoA, giving acetoacetate. Acetoacetate is reduced by NADH to D-(R)-3-hydroxybutyrate and slowly loses CO₂ to give acetone; these three are the ketone bodies. The liver makes them but cannot use them, because it lacks succinyl-CoA:3-oxoacid CoA-transferase, the enzyme that reactivates acetoacetate in heart, muscle and brain. In the cytosol HMG-CoA is reduced instead: HMG-CoA reductase uses two NADPH to turn the thioester into the primary alcohol of mevalonate, the committed step of cholesterol synthesis and the target of the statins.

Fatty acid synthesis: the same carbons, different chemistry

Acetyl-CoA carboxylase uses biotin, ATP and bicarbonate to make malonyl-CoA (shown as its N-acetylcysteamine model), the committed step. The malonyl group is moved to the phosphopantetheine thiol of the acyl carrier protein (ACP), and the growing acyl chain sits on a cysteine of the ketosynthase. The ketosynthase decarboxylates malonyl-ACP; the thioester enolate that results attacks the acyl-cysteine thioester, and the tetrahedral intermediate expels the cysteine, giving a 3-ketoacyl-ACP. Losing CO₂ makes this Claisen condensation strongly favourable, which a condensation of two acetyl thioesters is not. The ketoreductase then delivers hydride from NADPH and a tyrosine proton to give the D-(R)-3-hydroxyacyl-ACP, the opposite enantiomer of the β-oxidation intermediate; the dehydratase removes the α-proton with a histidine and loses the β-hydroxyl to give the trans-2-enoyl-ACP; and the enoyl reductase adds hydride from NADPH to the β-carbon. Seven cycles make palmitate, costing 7 ATP (for 7 malonyl-CoA) and 14 NADPH, in the cytosol rather than the mitochondrion.

Phosphoryl transfer from ATP

Nearly every enzymatic reaction of ATP is a nucleophilic attack on one of its three phosphorus atoms (ATP is shown), in line with the bond that breaks; the rare exceptions attack C5′ instead, as in S-adenosylmethionine synthesis. Attack at the γ-phosphorus transfers a phosphoryl group: kinases, ATP hydrolysis by an activated water, and adenylate kinase, in which a phosphate oxygen of AMP attacks ATP to make two ADP. Attack at the β-phosphorus transfers pyrophosphate and releases AMP: PRPP synthetase makes 5-phosphoribosyl 1-pyrophosphate this way, keeping the α-configuration of the ribose because the C1–O bond is not broken. Attack at the α-phosphorus transfers AMP and releases pyrophosphate: acyl-CoA synthetases. Nucleoside diphosphate kinase works in two halves (ping-pong): a histidine takes the γ-phosphoryl group from ATP, and the phosphohistidine passes it to any NDP. ATP hydrolysis has ΔG°′ ≈ −30.5 kJ/mol, but in a cell ΔG = ΔG°′ + RT ln([ADP][Pᵢ]/[ATP]) is about −50 kJ/mol because ATP is kept far above equilibrium. The adenylate energy charge, ([ATP] + ½[ADP]) / ([ATP] + [ADP] + [AMP]), is normally held between 0.8 and 0.95.

Making nucleotides and deoxynucleotides

Purines are assembled atom by atom on 5-phosphoribosylamine, which comes from PRPP, so the ring is built on the sugar; pyrimidines are made first as the free ring orotate, which orotate phosphoribosyltransferase then attaches to PRPP. Ribonucleotide reductase makes every deoxynucleotide by replacing the 2′-OH of an NDP with H. It uses radical chemistry: in the class I enzymes of humans and E. coli a stable tyrosyl radical next to a diiron centre generates a cysteine thiyl radical in the active site, which abstracts the 3′-hydrogen of the substrate. The 3′-radical lets the 2′-OH leave as water, two more cysteines deliver the electrons (forming a disulfide), and the 3′-hydrogen is returned at the end. Hydroxyurea destroys the tyrosyl radical and stops DNA synthesis. Thymidylate synthase converts dUMP into dTMP (both shown): a cysteine adds to C6, C5 attacks the methylene carbon of N⁵,N¹⁰-methylene-tetrahydrofolate, and the C5 hydrogen is then removed and a hydride from the folate reduces the exocyclic methylene to methyl, leaving dihydrofolate. 5-Fluoro-dUMP enters the same pathway but has no C5 hydrogen to lose, so it stays covalently bound.

What to carry forward

Lipid and nucleotide metabolism uses the same few reactions as the flask: thioester enolates for Claisen and aldol chemistry, hydride transfer to and from NAD(P) and flavins, conjugate additions and E1cB eliminations, and in-line attack on a chosen phosphorus of ATP. Track the carrier (CoA or ACP), the cofactor (FAD, NAD⁺ or NADPH) and the stereochemistry (L for breakdown, D for synthesis), and the energy bookkeeping follows from counting NADH, FADH₂, acetyl-CoA and phosphoanhydride bonds.

Problemas de prática (40)

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Desenhe o mecanismo 15

β-Oxidation of fatty acids

  • Acyl-CoA dehydrogenase: butyryl-CoA to trans-crotonyl-CoADraw the first step of β-oxidation: a glutamate removes the α-proton of butyryl-CoA while the β-hydrogen moves as hydride to N5 of FAD, giving trans-crotonyl-CoA and reduced flavin.
  • Enoyl-CoA hydratase: water adds to trans-crotonyl-CoA to give L-3-hydroxybutyryl-CoADraw the conjugate addition of glutamate-activated water to the β-carbon of trans-crotonyl-CoA and the protonation of the thioester enolate at the α-carbon that gives L-(S)-3-hydroxybutyryl-CoA.
  • 3-Hydroxyacyl-CoA dehydrogenase: NAD⁺ oxidises L-3-hydroxybutyryl-CoA to acetoacetyl-CoADraw the oxidation of the secondary alcohol of L-3-hydroxybutyryl-CoA to the ketone of acetoacetyl-CoA, with the hydroxyl proton going to a histidine and the C–H hydrogen going as hydride to C4 of NAD⁺.
  • Thiolase: retro-Claisen cleavage of 3-oxohexanoyl-CoADraw the attack of the thiolase cysteine on the ketone of 3-oxohexanoyl-CoA, the collapse that breaks the C2–C3 bond and expels the acetyl-CoA enolate, and the protonation of that enolate on carbon.
  • Thiolase: coenzyme A takes the butanoyl group off the enzymeDraw the transthioesterification in which coenzyme A attacks the butanoyl-cysteine thioester of thiolase and the tetrahedral intermediate releases the cysteine, giving butanoyl-CoA.

ATP and phosphoryl transfer

  • Adenylate kinase: AMP and ATP give two ADPDraw the in-line attack of an AMP phosphate oxygen on the γ-phosphorus of ATP that transfers one phosphoryl group and makes two ADP.
  • ATP hydrolysis: a glutamate-activated water attacks the γ-phosphorusDraw the in-line attack of a glutamate-activated water on the γ-phosphorus of ATP that releases ADP and hydrogen phosphate.
  • Nucleoside diphosphate kinase: ATP + GDP → ADP + GTP through phosphohistidineDraw the transfer of the γ-phosphoryl group of ATP to the active-site histidine of nucleoside diphosphate kinase, and its transfer from the phosphohistidine to GDP that gives GTP.
  • PRPP synthetase: pyrophosphoryl transfer from ATP to ribose 5-phosphateDraw the attack of the anomeric oxygen of α-D-ribose 5-phosphate on the β-phosphorus of ATP that transfers pyrophosphate and releases AMP, giving 5-phospho-α-D-ribosyl 1-pyrophosphate.

Fatty acid synthesis

  • Dehydratase: E1cB loss of water from D-3-hydroxybutyryl-ACPDraw the histidine-mediated removal of the α-proton of D-3-hydroxybutyryl-ACP and the E1cB loss of the protonated β-hydroxyl that gives trans-crotonyl-ACP.
  • Enoyl reductase: NADPH reduces trans-crotonyl-ACP to butyryl-ACPDraw the conjugate delivery of hydride from NADPH to the β-carbon of trans-crotonyl-ACP and the protonation of the resulting thioester enolate at the α-carbon, giving butyryl-ACP.
  • Ketoreductase: NADPH reduces acetoacetyl-ACP to D-3-hydroxybutyryl-ACPDraw hydride transfer from NADPH to the ketone of acetoacetyl-ACP with proton donation from a tyrosine, giving the D-(R)-3-hydroxyacyl-ACP.
  • Ketosynthase: decarboxylative Claisen condensation of malonyl-ACP with acetyl-enzymeDraw the decarboxylation of malonyl-ACP to the acetyl-ACP enolate, its addition to the acetyl-cysteine thioester of the ketosynthase, and the collapse that releases the cysteine and gives acetoacetyl-ACP.

Ketone bodies and HMG-CoA

  • HMG-CoA lyase: retro-aldol cleavage to acetoacetate and acetyl-CoADraw the deprotonation of the C3 hydroxyl of HMG-CoA, the retro-aldol cleavage that releases acetoacetate and the acetyl-CoA enolate, and protonation of that enolate on carbon.
  • HMG-CoA synthase: the acetyl-enzyme enolate adds to acetoacetyl-CoADraw the deprotonation of the acetyl-cysteine of HMG-CoA synthase by a glutamate and the addition of the resulting enolate to the ketone of acetoacetyl-CoA, with a histidine protonating the alkoxide.

Preveja o produto 8

Ketone bodies and HMG-CoA

  • The fruity-breath ketone bodyPredict the product of spontaneous decarboxylation of the β-keto acid acetoacetate.

β-Oxidation of fatty acids

  • What is left after one round?Predict the acyl-CoA that remains after one complete round of β-oxidation of a saturated acyl-CoA.
  • Put β-oxidation in orderOrder the activation step and the four reactions of a β-oxidation round by recognising each enzyme's substrate.
  • Getting past a cis double bondPredict the product of enoyl-CoA isomerase on the cis-Δ³ intermediate formed during oleate oxidation.

Fatty acid synthesis

  • Which 3-hydroxybutyryl intermediate does synthesis make?Predict the configuration of the 3-hydroxyacyl intermediate of fatty acid synthesis and contrast it with β-oxidation.

Nucleotide synthesis and deoxyribonucleotides

  • What does ribonucleotide reductase make from UDP?Predict which hydroxyl group ribonucleotide reductase removes from a ribonucleoside diphosphate.
  • Where the radical startsPredict which hydrogen the thiyl radical of ribonucleotide reductase abstracts from the ribose, and why that enables loss of the 2′-OH.
  • Thymidylate synthase: where the carbon goesPredict the product of thymidylate synthase and recognise that the folate supplies both the carbon and the reducing equivalents.

Classificar 2

Ketone bodies and HMG-CoA

  • A ketone body without a ketoneClassify the ketone bodies by functional group and identify the one that contains no ketone.

Nucleotide synthesis and deoxyribonucleotides

  • Ring first or sugar first?Classify the de novo purine and pyrimidine pathways by when the ribose phosphate is attached.

Comparar 2

Fatty acid synthesis

  • Follow C3 through a synthesis cycleRank the four acyl intermediates of a fatty acid synthesis cycle by the oxidation state of C3.

ATP and phosphoryl transfer

  • Rank phosphoryl donorsRank phosphorylated metabolites by their standard free energy of hydrolysis and relate the order to their structures.

Calcular 5

ATP and phosphoryl transfer

  • Adenylate energy chargeCalculate the adenylate energy charge of a cell from its ATP, ADP and AMP concentrations.
  • ΔG of ATP hydrolysis in a cellCalculate the actual free energy of ATP hydrolysis from ΔG°′ and cellular concentrations.

β-Oxidation of fatty acids

  • Acetyl-CoA from an odd-chain fatty acidCount the acetyl-CoA released when an odd-chain acyl-CoA is fully β-oxidised.
  • Net ATP from palmitateCalculate the net ATP yield of complete oxidation of palmitate from the products of β-oxidation.

Fatty acid synthesis

  • NADPH cost of palmitateCalculate the NADPH consumed by fatty acid synthase to make one palmitate from acetyl-CoA and malonyl-CoA.

Encontre o erro 1

  • Find the error: synthesis is β-oxidation in reverseDiagnose the flaw in treating fatty acid synthesis as β-oxidation run backwards.

Interprete as evidências 1

  • Reading an EPR result for hydroxyureaInterpret the loss of the tyrosyl radical signal as the mechanism by which hydroxyurea stops ribonucleotide reductase.

Planeje uma síntese 2

Fatty acid synthesis

  • What does acetyl-CoA carboxylase need?Choose the cofactor and co-substrates that a biotin-dependent carboxylase uses to make malonyl-CoA.

Ketone bodies and HMG-CoA

  • Where to block cholesterol synthesisChoose the enzyme whose inhibition most selectively lowers cholesterol synthesis, using the committed step of the mevalonate pathway.

Explique por quê 4

β-Oxidation of fatty acids

  • Why fatty acids need carnitineExplain why long-chain acyl groups cross the inner mitochondrial membrane as acylcarnitines.

Nucleotide synthesis and deoxyribonucleotides

  • Why 5-fluoro-dUMP kills thymidylate synthaseExplain why 5-fluoro-dUMP forms a dead-end covalent complex with thymidylate synthase.

Ketone bodies and HMG-CoA

  • Why the liver does not burn its ketone bodiesExplain which missing enzyme makes the liver a net exporter of ketone bodies.

Fatty acid synthesis

  • Stopping a futile cycleExplain how malonyl-CoA prevents newly made fatty acids from being oxidised at the same time.

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