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Chapter 16

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CHY 205
Mario Estable

Chapter 16: The Citric Acid cycle -The oxidation of pyruvate to H2O & CO2 in aerobic conditions is called respiration, more specifically cellular respiration which refers to molecular processes by which cells consume O2 & produce CO2. This occurs in 3 stages: 1) Acetyl-coA production: Organic fuel molecules, eg. glc, FA & some aa, are oxidized to form acetyl-coA. Glc to pyruvate via glycolysis & pyruvate to acetyl-coA via pyruvate dehydrogenase complex. 2) Acetyl-coA oxidation: Acetyl groups are fed into CAC which enzymatically oxidizes them to CO2, the energy released is conserved in reduced e- carriers NADH & FADH2. This stage generates more NADH, FADH2, 1 GTP/ATP. 3) E- transfer & oxidative phosphorylation: The reduced coenzymes are themselves oxidized giving up H+ & e- which are transferred to O2, the final e- acceptor via the respiratory chain. This e- flow drives the production of ATP in what is known as oxidative phosphorylation. Production of Acetyl-coA (activated acetate) -Before entering the CAC, the C skeletons of sugars & FA are degraded to the acetyl group of acetyl-coA. Here we cous on how pyruvate derived from glc & other sugars by glycolysis is oxidized to acetyl-coA & CO2 by the pyruvate dehydrogenase complex (PDHC), a cluster of enzymes that have multiple copies of each of 3 enzymes, are located in the mitochondria of eukaryotes & cytosol of bacteria. The net reaction is decarboxylation of pyruvate to acetyl group attached to co-A which can enter the CAC & yield energy, it can also be used to synthesize storage lipids. This is known as oxidative decarboxylation which req 5 coenzymes & is catalyzed by the PDHC & irreversible. The NADH formed in this rxn gives up a :H- to the ETC which carries 2 e- to O, NO3- or SO42-. The transfer of e-s from NADH to O ultimately generates 2.5 ATP/e- pair. -The 5 coenzymes are TPP, FAD, coA/coA-SH, NAD & lipoate. PDHC is a large multienzyme complex (Mr= 7.8x10^6 Da) which contains 3 distinct enzymes: pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2) & dihydrolipoyl dehydrogenase (E3). Part of the complex are 2 regulatory pro, protein kinase & phosphoprotein phosphatase. The short dist b/w their catalytic sites allows channelling of substrates from catalytic site to another & this channelling minimizes side rxns b/c intermediates never leave the enzyme surface. The activity of the complex is subject to regulation by [ATP]. E1) pyruvate dehydrogenase + TPP (from thiamine) E2) dihydrolipyl transacetylase + lipoate, coA-SH (from patothenate) E3) dihydrolipoyl dehydrogenase + FAD (from riboflavin), NAD (from niacin) *The lipoyl group of lipoyllysine occurs in oxidized (disulfide) & reduced (dithiol) forms & acts as a carrier of both H & acetyl group. -Sequence of events in Pyruvate: 1. Decarboxylation of pyruvate to hydroxyethyl TPP: Pyruvate reacts w/ bound TPP of E1 undergoing decarboxylation to hydroxyethyl TPP. This is the slowest step & therefore the rate-limiting step & also the pt at which the PDHC complex exercises its substrate specificity. 2. Reduction of lipoyllysine to acyllipoyllysine: E1 carries out the transfer of 2 e-s & acetyl group from TPP to the oxidized form of the lipoyllysyl group of the core enzyme, E2, to form the acetyl thioester of the reduced lipoyl group, acyllipoyllysine. 3. Transesterification of coA-SH to acetyl-coA: the –SH group of coA replaces the – SH group of E2 to yield acetyl co-A & the fully reduced (dithiol) form of the lipoyl group. 4. Reoxidation of the lipoyllysine: E3 promotes the transfer of 2 H atoms from the reduced lipoyl groups of ED to the FAD prosthetic group of E3, restoring the oxidized form of the lipoyllysyl group of E2. 5. Regeneration of the oxidized FAD cofactor: The reduced FADH2 of E3 transfers a :H- to NAD+ forming NADH. The enzyme complex is now ready for another catalytic cycle. *The organization of the PDHC is very similar to enzyme complexes that catalyze the oxidation of alpha-ketoglutarate & branched-chain alpha-keto acids. Sequence of Events in the CAC *In each turn of the cycle, 1 acetyl-coA enters & 2CO2 leaves, 1 oxaloacetate is used to form citrate & 1 oxaloacetate is generated. There is no net removal of oxaloacetate & in fact it is present in very low [ ] in cells. 1. Claisen Condensation (Formation of citrate): condensation of acetyl-coA (2C) w/ oxaloacetate (4C) to form citrate (6C) catalyzed by citrate synthase. Acetyl-coA donates its acetyl group to oxaloacetate to form citrate. Highly exergonic & irreversible b/c of the hydrolysis of high energy thioester intermediate. The coA liberated is recycled to participate in oxidative decarboxylation of another pyruvate by the PDHC. 2. Isomerization via dehydration followed by hydration (Formation of isocitrate via cis- Aconitate): Aconitase, aka aconitate hydratase, catalyzes the reversible transformation of citrate (6C) to isocitrate (6C) via intermediary formation of cis- aconitate (6C) which normally doesn’t dissociate from the active site. Aconitase can promote the reversible addition of H2O to the = bond of enzyme-bound cis-aconitate in 2 ways, one leading to citrate & leading to isocitrate: even though the equilibrium mix at pH 7.4 & 25°C contains <10% isocitrate, cell rxn continues to the right b/c isocitrate is rapidly consumed in the enxt step lowering its steady-state [ ]; aconitase has an Fe-S center which acts both in the binding of the substrate at the active site & catalytic addition/removal of H2O. 3. Oxidative decarboxylation (irreversible oxidation of isocitrate to alpha-ketoglutarate & CO2): Isocitrate dehydrogenase catalyzes oxidative decarboxylation of isocitrate (6C) to form alpha-ketoglutarate (5C). Mn2+ in the active site interacts w/ oxalosuccinate which is formed transiently & doesn’t leave the binding site until decarboxylation converts it to alpha-ketoglutarate. There are 2 forms of isocitrate dehydrogenase, one req NAD+ as an e- acceptor & the other req NADP+. 4. Oxidative decarboxylation (irreversible oxidation of alpha-ketoglutarate to succinyl- coA & CO2): alpha-ketoglutarate (5C) is oxidized to succinyl-coA (4C) & CO2 by the action of alpha-ketoglutarate dehydrogenase complex where NAD+ serves as the e- acceptor & coA as the carrier for succinyl. This complex closely resembles PDHC, it also incl 3 enzymes homologous to E1, E2 & E3 as well as enzyme-bound TPP, lipate, FAD, NAD & co-A. 5. Substrate-level phosphorylation (reversible conversion of succinyl-coA (4C) to succinate): energy released from the breakage of the bond b/w succinyl & coA is used to drive the synthesis of a phosphoanhydride bond in GTP/ATP forming succinate (4C) in the process catalyzed by succinyl-coA synthetase, aka succinic thiokinase. The enzyme becomes phosphorylated & the phosphoryl group is transferred to GDP to form GTP which donates its gamma phosphoryl to ADP to form ATP in a reversible rxn catalyzed by nucleoside diphosphate kinase. 6. Dehydrogenation (reversible oxidation of succinate to fumarate): The succinate (4C) formed from succinyl-coA is oxidized to fumarate (4C) by the flavopro succinate dehydrogenase. E- pass from succinate through FAD & Fe sulphur centers in the enzyme before entering ETC in mitochondrial membrane. E- flow
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