Arrow Lab

Mecanismos de Bioquímica · Enzymes and cofactors

Cofatores e coenzimas

Uma lição resolvida gratuita e 40 problemas de prática em que você mesmo desenha cada seta curva.

Travou em cofatores e coenzimas? Escaneie o problema.

Tire uma foto da reação da sua lista ou do livro e desenhe o mecanismo com cada seta corrigida. 1 scan grátis agora, sem cadastro; mais 1 com uma conta gratuita.

Escaneie sua lição de graça

O que você vai aprender

  • Draw hydride transfer between C4 of NADH or NAD⁺ and a substrate, including the active-site acid or base that moves the proton.
  • Explain how a flavin accepts a hydride at N5 and why it can hand electrons on one at a time.
  • Use the pyridoxal phosphate Schiff base as an electron sink to draw transimination, transamination and decarboxylation.
  • Draw thiamine ylide formation and α-keto acid decarboxylation through the enamine (Breslow) intermediate.
  • Follow acyl and carboxyl groups as they move through lipoamide, coenzyme A thioesters and carboxybiotin.

Lição gratuita

O texto da lição resolvida. No Arrow Lab, cada etapa vem com as estruturas e as setas desenhadas.

Ler a aula gratuita (7 partes)

Cofactors are borrowed functional groups

Amino acid side chains can act as acids, bases and nucleophiles, but none of them can carry a hydride, stabilise a carbanion at the α-carbon of an amino acid, or hold an acyl group between two active sites. Enzymes borrow these abilities from cofactors, most of which are made from vitamins: NAD⁺ from niacin, FAD from riboflavin, pyridoxal phosphate (PLP) from vitamin B₆, thiamine diphosphate (TPP) from vitamin B₁, coenzyme A from pantothenate, and biotin, which is itself a vitamin (B₇). Each cofactor has a small reactive core; the rest of the molecule (ribose, phosphates, adenine) is a handle the enzyme grips. In this topic the cores are drawn alone: NAD⁺ as 1-methylnicotinamide, flavins as lumiflavin, PLP as an N-methylpyridinium aldehyde, TPP as 3,4-dimethylthiazolium, coenzyme A as N-acetylcysteamine, lipoamide as a methyl-1,2-dithiolane and biotin as its ureido ring. The arrows on these cores are exactly the arrows on the real cofactors.

NAD⁺ and NADH carry a hydride at C4

NAD⁺ is a pyridinium ion. A hydride (H⁻, two electrons and a proton) adds to C4, opposite the ring nitrogen, and gives NADH, a 1,4-dihydropyridine; the ring loses its aromaticity and its positive charge. The reverse, hydride loss from C4 of NADH, is pushed by the nitrogen lone pair and restores the aromatic ring. Dehydrogenases pair this hydride move with a proton move: in lactate dehydrogenase a histidinium protonates the carbonyl oxygen of pyruvate as the hydride arrives, and in glutamate dehydrogenase a lysine frees the amino lone pair, which then pushes the α-hydrogen of glutamate onto NAD⁺ as a hydride, giving an iminium ion. Each enzyme uses one face of C4: lactate, malate and alcohol dehydrogenases transfer the pro-R hydrogen (A-side). NADH absorbs at 340 nm (ε ≈ 6220 M⁻¹ cm⁻¹) and NAD⁺ does not, so these reactions are followed by the absorbance at 340 nm. The standard potential of NAD⁺/NADH is about −0.32 V, low enough that NADH reduces carbonyls and, through the electron-transport chain, O₂.

Flavins: hydride in, electrons out one at a time

The isoalloxazine ring of FAD and FMN (drawn here as lumiflavin) accepts a hydride at N5. The C4a=N5 and C10a=N1 π bonds shift so the extra pair ends on N1, giving the reduced flavin anion FADH⁻ (protonation at N1 gives FADH₂). D-amino acid oxidase removes the α-hydrogen of a D-amino acid as hydride this way, and acyl-CoA dehydrogenase removes the α-proton with a glutamate base while the β-hydrogen goes to N5 as hydride, making a trans C=C bond. The same arrows run backwards when a reduced flavin gives a hydride from N5 to NAD⁺, as in the E3 enzyme of pyruvate dehydrogenase. Flavins differ from NAD⁺ because the one-electron intermediate, the semiquinone, is stable: a reduced flavin can pass its two electrons one at a time to iron–sulfur clusters, ubiquinone or O₂. Flavins are usually bound tightly or covalently, and the protein tunes their potential; free FAD/FADH₂ is about −0.22 V.

PLP: the Schiff base is an electron sink

In the resting enzyme the PLP aldehyde is bound to a lysine as a protonated imine (the internal aldimine). An incoming amino acid displaces the lysine by transimination, giving the external aldimine shown. Now every bond to the α-carbon is labile, because the electrons of a breaking Cα bond flow through the protonated imine into the pyridinium ring, whose nitrogen takes up the pair: this delocalised species is the quinonoid intermediate. The enzyme chooses which bond breaks by holding that bond perpendicular to the plane of the ring and imine, where it overlaps the π system. Breaking Cα–H and reprotonating at C4′ converts the aldimine into a ketimine; hydrolysis then releases an α-keto acid and pyridoxamine phosphate (PMP), which is transamination. Breaking Cα–CO₂⁻ and reprotonating at Cα gives an amine (decarboxylation), for example GABA from glutamate and dopamine from L-DOPA.

Thiamine: a carbanion that decarboxylates α-keto acids

C2 of the thiazolium ring of TPP sits between a positively charged nitrogen and a sulfur. Its C–H proton is removed (pKa about 18 in water; in the enzyme by the 4′-imino group of the cofactor's own aminopyrimidine) to give an ylide, a carbanion in the plane of the ring. The ylide adds to the ketone carbonyl of an α-keto acid such as pyruvate. The adduct now has its carboxylate β to the C=N⁺ of the thiazolium, so CO₂ leaves just as from a β-keto acid, and the electrons are stored in the enamine called the Breslow intermediate. Pyruvate decarboxylase protonates it and releases acetaldehyde; pyruvate dehydrogenase instead lets it attack lipoamide, which oxidises the two-carbon unit to an acetyl group.

Carriers of acyl and carboxyl groups

Lipoamide is a cyclic disulfide on a long lysine arm. Attacked by the Breslow enamine, it is reduced to a dithiol that carries the acetyl group as a thioester to coenzyme A; the dithiol is then reoxidised by disulfide exchange with the FAD enzyme E3, and FADH₂ reduces NAD⁺. Coenzyme A ends in a thiol; its thioesters are good acyl donors (sulfur shares its lone pairs poorly with the C=O, and thiolate is a good leaving group) and have unusually acidic α-hydrogens. Acetyl-CoA synthetase makes them from an acyl adenylate. Biotin is attached to a lysine and carries CO₂ on the N1′ nitrogen of its ureido ring. ATP activates bicarbonate as carboxyphosphate, which carboxylates biotin; carboxybiotin then releases CO₂ next to an enolate of the acceptor (pyruvate, acetyl-CoA or propionyl-CoA), which captures it.

What to carry forward

Find the cofactor's reactive core and treat it as an ordinary functional group: NAD⁺ is a pyridinium that takes hydride at C4, a flavin takes hydride at N5 and gives electrons back one at a time, the PLP imine and the thiazolium C=N⁺ are electron sinks that make α-carbanions possible, and thiols (lipoamide, coenzyme A) and biotin carry acyl and carboxyl groups between active sites.

Problemas de prática (40)

Praticar é grátis com uma conta.

Desenhe o mecanismo 15

Coenzyme A and biotin

  • Acetyl-CoA synthetase: acetate is activated as acetyl adenylate and transferred to coenzyme ADraw adenylation of acetate at the α-phosphorus of ATP with loss of pyrophosphate, then thiolysis of the acyl adenylate by coenzyme A to give acetyl-CoA and AMP.
  • Pyruvate carboxylase: carboxybiotin carboxylates pyruvate to oxaloacetateDraw decarboxylation of carboxybiotin, deprotonation of pyruvate by the biotin anion and capture of CO₂ by the pyruvate enolate to give oxaloacetate.
  • Pyruvate dehydrogenase E2: the acetyl group moves from lipoamide to coenzyme ADraw transthioesterification from S-acetyldihydrolipoamide to coenzyme A through a tetrahedral intermediate, with histidine as the base and acid.

NAD⁺ and flavin redox

  • D-Amino acid oxidase: hydride transfer from D-alanine to the flavin N5Draw the hydride transfer from the α-carbon of D-alanine to N5 of an oxidised flavin, with the amino lone pair forming the imine and the flavin electrons ending on N1.
  • Dihydrolipoamide dehydrogenase (E3): reduced FAD passes a hydride to NAD⁺Draw hydride transfer from N5 of a reduced flavin anion to C4 of NAD⁺, regenerating the oxidised flavin and forming NADH.
  • Glutamate dehydrogenase: NAD⁺ oxidises L-glutamate to 2-iminoglutarateDraw deprotonation of the α-ammonium group of L-glutamate and the hydride transfer from its α-carbon to C4 of NAD⁺ that forms 2-iminoglutarate.
  • Lactate dehydrogenase: NADH reduces pyruvate to L-lactateDraw hydride transfer from C4 of NADH to the ketone carbon of pyruvate, with the histidinium proton delivered to the carbonyl oxygen, giving L-lactate and NAD⁺.

Thiamine and lipoamide

  • Pyruvate dehydrogenase E1: the enamine transfers an acetyl group to lipoamideDraw the attack of the thiamine enamine on the lipoamide disulfide and the collapse that releases the ylide and leaves S-acetyldihydrolipoamide.
  • Pyruvate dehydrogenase E3: dihydrolipoamide is reoxidised by thiol–disulfide exchangeDraw the two thiol–disulfide exchanges that transfer two electrons from dihydrolipoamide to the E3 cystine and close the lipoamide dithiolane ring.
  • Pyruvate decarboxylase: decarboxylation through the Breslow enamine and release of acetaldehydeDraw loss of CO₂ from lactyl-ThDP to the enamine, protonation of the enamine carbon and release of acetaldehyde with regeneration of the ylide.
  • Pyruvate decarboxylase: the thiamine ylide adds to pyruvateDraw formation of the thiazolium ylide by the iminopyrimidine base, its addition to the ketone carbonyl of pyruvate and protonation of the alkoxide to give lactyl-ThDP.

Pyridoxal phosphate

  • Transamination: the alanine external aldimine becomes a ketimineDraw removal of the α-hydrogen from the alanine–PLP aldimine to form the quinonoid intermediate, then protonation at C4′ to form the ketimine.
  • Glutamate decarboxylase: PLP-dependent formation of GABADraw loss of CO₂ from the PLP–glutamate external aldimine into the quinonoid intermediate and protonation at Cα that gives the GABA aldimine.
  • Transamination: hydrolysis of the ketimine to pyruvate and PMPDraw the hydrolysis of the PLP ketimine through a carbinolamine to pyruvate and pyridoxamine phosphate, with the lysine as the proton shuttle.
  • PLP transimination: L-alanine replaces the active-site lysineDraw transimination of the PLP internal aldimine by L-alanine through the gem-diamine to the external aldimine, releasing the lysine amine.

Preveja o produto 5

Pyridoxal phosphate

  • Product of aspartate transaminationPredict the keto acid released when L-aspartate gives its amino group to PLP.
  • Product of DOPA decarboxylasePredict the amine formed when a PLP-dependent decarboxylase acts on L-DOPA.

NAD⁺ and flavin redox

  • Which ring is NADH?Identify the 1,4-dihydropyridine formed when NAD⁺ accepts a hydride at C4.

Thiamine and lipoamide

  • Order the pyruvate dehydrogenase stepsOrder the cofactor events of the pyruvate dehydrogenase complex from pyruvate to NADH.

Coenzyme A and biotin

  • Product of propionyl-CoA carboxylasePredict where biotin-dependent carboxylation places CO₂ on propionyl-CoA.

Classificar 3

Coenzyme A and biotin

  • Where biotin carries CO₂Identify the atom of biotin that carries CO₂ in carboxybiotin.
  • The vitamin inside coenzyme AClassify coenzyme A by the vitamin it is built from and distinguish it from the vitamin sources of other cofactors.

Thiamine and lipoamide

  • Which form of thiamine is the nucleophile?Identify the thiazolium ylide as the nucleophilic form of thiamine diphosphate.

Comparar 2

NAD⁺ and flavin redox

  • Rank biological reductantsRank the reduced partners of biological redox couples by reducing strength from their standard reduction potentials.

Coenzyme A and biotin

  • Rank α-hydrogen acidityRank the α-C–H acidity of an amide, an ester, a thioester and a ketone and relate it to coenzyme A chemistry.

Calcular 3

Coenzyme A and biotin

  • Energetics of acetyl-CoA synthetaseCalculate the standard free-energy change for acetate activation by acetyl-CoA synthetase coupled to pyrophosphate hydrolysis.

NAD⁺ and flavin redox

  • NADH concentration from A₃₄₀Calculate an NADH concentration from its absorbance at 340 nm with the Beer–Lambert law.
  • Free energy of NADH oxidation by O₂Calculate ΔG°′ for oxidation of NADH by O₂ from standard reduction potentials.

Encontre o erro 1

  • Find the flaw in a decarboxylation mechanismDiagnose why a decarboxylation drawn directly from the free amino acid cannot occur.

Interprete as evidências 1

  • Read a lactate dehydrogenase assayConvert the rate of absorbance decrease at 340 nm in a lactate dehydrogenase assay into nanomoles of NADH oxidised per minute.

Trabalhe de trás para frente 2

Pyridoxal phosphate

  • Which keto acid becomes phenylalanine?Work backwards from an amino acid to the α-keto acid that an aminotransferase converts into it.

Thiamine and lipoamide

  • Which α-keto acid gives 3-methylbutanal?Work backwards from an aldehyde to the α-keto acid that a ThDP decarboxylase converts into it.

Planeje uma síntese 1

  • Choose the cofactor for a C–C dehydrogenationChoose the cofactor an enzyme uses to oxidise a CH₂–CH₂ unit to a trans C=C bond, using reduction potentials.

Explique por quê 7

Thiamine and lipoamide

  • Why α-keto acids need thiamineExplain why α-keto acids need thiamine diphosphate to decarboxylate while β-keto acids decarboxylate on their own.
  • How arsenite stops pyruvate dehydrogenaseExplain why arsenite inhibits the lipoamide-dependent 2-oxo acid dehydrogenases.
  • Why C2 of thiamine can be deprotonatedExplain what stabilises the thiamine ylide and how the enzyme forms it at pH 7.

NAD⁺ and flavin redox

  • Why flavins talk to iron–sulfur clustersExplain why flavins, but not NAD⁺, can pass electrons to one-electron carriers.

Pyridoxal phosphate

  • How does a PLP enzyme pick the bond?Explain how a PLP enzyme selects which bond to the α-carbon breaks in the external aldimine.
  • What PLP does for the α-carbonExplain how the PLP Schiff base makes the bonds to the α-carbon of an amino acid labile.

Coenzyme A and biotin

  • Why biology stores acyl groups as thioestersExplain why a coenzyme A thioester transfers its acyl group more readily than an oxygen ester.

Travou em cofatores e coenzimas? Escaneie o problema.

Tire uma foto da reação da sua lista ou do livro e desenhe o mecanismo com cada seta corrigida. 1 scan grátis agora, sem cadastro; mais 1 com uma conta gratuita.

Escaneie sua lição de graça

Antes deste tópico