Covalent Modification and Mechanisms of the Eukaryotic Pyruvate Dehydrogenase Complex

1. Lecture 26 • TCA cycle

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3. Figure 21-17b Factors controlling the activity of the PDC.(b) Covalent modification in the eukaryotic complex. Page 781

4. Control by phosporylation/dephosphorylation • Occurs only in eukaryotic complexes • The E2 subunit has both a pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase that act to regulate the E1 subunit. • Kinase inactivates the E1 subunit. Phosphatase activates the subunit. • Ca2+ is an important secondary messenger, it enhances phosphatase activity.

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6. Citric acid cycle: 8 enzymes • Oxidize an acetyl group to 2 CO2 molecules and generates 3 NADH, 1 FADH2, and 1 GTP. • Citrate synthase: catalyzes the condensation of acetyl-CoA and oxaloacetate to yield citrate. • Aconitase: isomerizes citrate to the easily oxidized isocitrate. • Isocitrate dehydrogenase: oxidizes isocitrate to the -keto acid oxalosuccinate, coupled to NADH formation. Oxalosuccinate is then decarboxylated to form -ketoglutarate. (1st NADH and CO2). • -ketoglutarate dehydrogenase: oxidatively decarboxylates -ketoglutarate to succinyl-CoA. (2nd NADH and CO2). • Succinyl-CoA synthetase converts succinyl-CoA to succinate. Forms GTP. • Succinate dehydrogenase: catalyzes the oxidation of central single bond of succinate to a trans double bond, yielding fumarate and FADH2. • Fumarase: catalyzes the hydration of the double bond to produce malate. • Malate dehydrogenase: reforms OAA by oxidizing 2ndary OH group to ketone (3rd NADH)

7. Citric acid cycle 3NADH + FADH2 + GTP + CoA + 2CO2 3NAD+ + FAD + GDP + Pi + acetyl-CoA 3NADH + FADH2 are oxidized by the electron transport chain and drive ATP synthesis.

8. Citrate synthase: reaction 1 • Catalyzes the condensation of acetyl-CoA and oxaloacetate to form citrate. • Oxaloacetate has to bind to the enzyme before acetyl-CoA. • Oxaloacetate binds to the enzyme causing a conformational shift that opens the acetyl-CoA binding site. (induced fit) • Reaction mechanism is a mixed aldol-Claisen ester condensation (acid-base catalysis). • Acetyl forms an enol intermediate. • Three important amino acids: His274, Asp375, His320 • The formation of enolate form of acetyl-CoA is the rate-limiting step. Asp375 acts as a general base to remove a proton from the methyl group of the acetyl-CoA. His 274 is hydrogen bonded to acetyl-CoA. • Citryl-CoA is formed in a second acid-base catalyzed reaction step. Acetyl-CoA enolate form attacks oxaloacetate. • Citryl-CoA is hydrolyzed to citrate and CoA. Stereospecific reactions (acetate onlly forms citrate’s pro-S carboxymethyl group.

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10. Aconitase: reaction 2 • Catalyzes the reversible isomerization of citrate and isocitrate with cis-aconitate as an intermediate. • Citrate is prochiral so aconitase can distinguish between citrate’s pro-R and pro-S carboxymethyl groups. • Has a covalently bound [4Fe-4S] iron-sulfur cluster. • Fea atom coordinates with the OH group of citrate • The iron-sulfur cluster does not perform a redox reaction but instead is able to stabilize the ligand-substrate complex. • Second stage of the reaction rehydrates cis-aconitate’s double bond in a stereospecific trans addition to form only the 2R,3S isocitrate form.

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12. Isocitrate dehydrogenase: reaction 3 • Catalyzes the oxidiation of isocitrate to form a-ketoglutarate • 1st reaction to produce NADH and CO2. • Activated by AMP and ADP • Inhibited by NADH and NADPH • Competitively bind to the NAD+ binding site. • Requires Mn2+ or Mg2+ cofactor. • Mechanistically-oxidize to the b-keto acid. • 2 forms of the enzyme • Mitochondrial form is NAD+ dependant [ADP] • E. coli, mitochondrial, cytoplasmic forms NADP+ dependant.

13. Figure 21-21 Probable reaction mechanism of isocitrate dehydrogenase. Page 785

14. -ketoglutarte dehydrogenase complex • Catalyzes the oxidiation and decarboxylation of -ketoglutarate to produce succinyl-CoA. • Consists of -ketoglutarte dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase (E3). • Mechanistically resembles PDC. • 2nd reaction to produce NADH and CO2. • 5 coenzymes (TPP, lipoic acid, CoA, FAD, NAD+) • Product inhibition (Succinyl-CoA), NADH

15. Reaction 1: -ketoglutarate dehydrogenase R -ketoglutarate O (+) N CH3 C-O- (-) C=O R H+ O S CH2 (+) C-O- E1 N CH3 CH2 CH2 CH2 C-OH C-O- CH2 P-P-O O S CH2 E1 TPP (ylid form) CH2 CO2 C-O- CH2 O P-P-O

16. S S E2 Reaction 2: Dihydrolipoyl transacetylase (E2) R + N CH3 - H+ C-OH CH2 S CH2 E1 CH2 C-O- CH2 O P-P-O Lipoamide-E2 -hydroxy--carboxy-propyl TPP-E1 complex -hydroxy group carbanion attacks the lipoamide disulfide causing the reduction of the disulfide bond

17. Dihydrolipoyl transacetylase (E2) R S + N CH3 H+ C-O-H CH2 E2 HS S CH2 E1 CH2 C-O- CH2 O The TPP is eliminated to form succinyl -dihydrolipoamide and regenerate E1 P-P-O -hydroxy--carboxy-propyl TPP-E1 complex

18. Dihydrolipoyl transacetylase (E2) R O C-O- + N CH3 CH2 - CH2 O S C E1 CH2 S CH2 P-P-O HS TPP-E1 complex Back to reaction 1 E2 Succinyl-dilipoamide-E2

19. O C-O- CH2 CH2 O C Reaction 3: Dihydrolipoyl transacetylase (E2) O CoA-S C CH2-CH2-COO- Succinyl-CoA + S HS CoA-SH HS HS E2 E2 Succinyl-dilipoamide-E2 dihydrolipamide-E2 E2 catalyzes the transfer of the succinyl group to CoA via a transesterification reaction where the sulfhydryl group of CoA attacks the acetyl group of the acetyl dilipoamide-E2 complex.

20. S HS S E2 HS E2 Reaction 4: Dihydrolipoyl dehydrogenase (E3) FAD FAD SH S SH S E3 reduced E3 oxidized + + E3 is oxidized and catalyzes the oxidation of dihydrolipoamide completing the cycle of E2.

21. FAD FADH2 SH S SH Reaction 5: Dihydrolipoyl dehydrogenase (E3) FAD S S S E3 oxidized NADH + H+ NAD+ E3 is oxidized by the enzyme bound FAD which is reduced to FADH2. This reduces NAD+ to produce NADH.

22. Succinyl-CoA Synthetase • Hydrolyzes the “high-energy” succinyl-CoA with the coupled synthesis of a “high-energy” nucleoside triphosphate. • In mammals, GTP • In bacteria and plants, ATP.

23. Succinyl-CoA Synthetase Succinyl-P Pi CoASH Enz-His Succinyl-CoA • Mechanistically: Succinate Enz-His Enz-His-P GTP Mg++ Enz-His-P Succinate GDP

24. Succinyl-CoA Synthetase Succinyl-P Pi CoASH Enz-His Succinyl-CoA • Mechanistically: Succinate Enz-His Enz-His-P GTP Mg++ Enz-His-P Succinate GDP

25. Figure 21-22a Reactions catalyzed by succinyl-CoA synthetase. Formation of succinyl phosphate, a “high-energy” mixed anhydride. Page 787

26. Figure 21-22b Reactions catalyzed by succinyl-CoA synthetase. Formation of phosphoryl–His, a “high-energy” intermediate. Page 787

27. Figure 21-22c Reactions catalyzed by succinyl-CoA synthetase. Transfer of the phosphoryl group to GDP, forming GTP. Page 787

28. Succinate dehydrogenase • Only makes the trans-fumarate. • Donates electrons directly into complex II of the respiratory chain (ubiquinone (Q)). • If the respiratory chain is inhibited, FAD is unable to accept electrons and TCA cycle stops. • Inhibited by OAA, activated by coenzyme Q (part of electron tranport chain).

29. Figure 21-23 Covalent attachment of FAD to a His residue of succinate dehydrogenase. Page 787

30. FADH2 COO- H-C-H Succinate dehydrogenase H-C-H COO- Succinate H-C-COO- -OOC-C-H Fumarate Succinate dehydrogenase Electron transport chain FAD

31. Succinate dehydrogenase • Catalyzes the stereospecific dehydrogenation of succinate to fumurate. • Enzyme strongly inhibited by malonate (competitive inhibitor). • Contains an FAD-electron acceptor. • FAD functions to oxidize alkanes to alkenes (vs. NAD+ which oxidizes alcohols to aldehydes and ketones). • FAD covalently linked to His from enzyme.

32. Fumarase (fumarate hydratase) • Catalyzes the stereospecific dehydrogenation of succinate to fumurate. • Only catalyzes the trans-fumarate • Competitively inhibited by maleate (cis double-bond).

33. H-C-COO- -OOC-C-H Fumarate Fumarase COO- H2O HO-C-H H-C-H Fumarase COO- S-malate

34. COO- O=C-H H-C-H COO- Oxaloacetate Malate dehydrogenase • Catalyzes the final reaction of the citric acid cycle-regeneration of oxaloacetate. • Oxidizes S-malate’s OH group to a ketone in an NAD+ dependent reaction. • Produces NADH. NADH COO- NAD+ HO-C-H Malate dehydrogenase H-C-H COO- S-malate

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36. Total (PDH and TCA) PDH NAD+ + pyruvate + CoA NADH + acetyl-CoA + CO2 TCA 3NADH + FADH2 + GTP + CoA + 2CO2 3NAD+ + FAD + GDP + Pi + acetyl-CoA Pyruvate 4NAD+ FAD GDP + Pi 3CO2 4NADH FADH2 GTP NADH DH 12ATP Complex II 2ATP 1ATP Nucleoside diphosphokinase

37. Figure 21-25 Regulation of the citric acid cycle. Page 791

38. Regulation of citric acid cycle • Rate-controlling enzymes: citrate synthase, isocitrate dehydrogenase, -ketoglutarate dehydrogenase. • Regulated by substrate availability, product inhibition and inhibition by other cycle intermediates (generally simpler than glycolysis). • Citrate synthase-inhibited by citrate, -KG, succ-CoA, NADH, activated by OAA and CoASH. • Isocitrate dehydrogenase-Requires AMP/ADP Activated by Ca2+, inhibited by NADPH or NADH • -ketoglutarate dehydrogenase-inhibited by Succ-CoA, NADH, ATP. Activated by Ca2+ • Pyruvate dehydrogenase-inhibited by NADH and acetyl-CoA

39. Figure 21-26 Amphibolic functions of the citric acid cycle. Page 793

40. Pathways that use citric acid cycle intermediates Reactions that utilize intermediates of TCA cycle are called cataplerotic reactions • Gluconeogenesis-in cytosol uses OAA. In the mitochondria uses malate (transported across the membrane). • Lipidbiosynthesis-requires acetyl-CoA. Transported across the membrane by the breakdown of citrate. ATP + citrate + CoA ADP + Pi + oxaloacetate + acetyl-CoA • Amino acid biosynthesis-can use -ketoglutarate to form glutamic acid in a reductive amination reaction (uses NAD+ or NADP+ depending on enzyme) -ketoglutarate + NAD(P)H + NH4+glutamate + NAD(P)+ + H2O

41. Pathways that use citric acid cycle intermediates 3. Amino acid biosynthesis-can also use -ketoglutarate and oxaloacactate in transamination reactions -ketoglutarate + alanineglutamate + pyruvate oxaloacetate + alanineaspartate + pyruvate • Porphyrin biosynthesis- utilizes succinyl-CoA • Complete oxidation of amino acids - amino acids first converted to PE by PEPCK

42. Pathways that make citric acid cycle intermediates Reactions that replenish intermediates of TCA cycle are called anaplerotic reactions Pyruvate carboxylase- produces oxaloacetate Pyruvate + CO2 + ATP + H2O oxaloacetate + ADP + Pi Degradative pathways generate TCA cycle intermediates • Oxidation of odd-chain fatty acids generates succinyl-CoA • Ile, Met, Val generate succinyl-CoA • Transamination and deamination of amino acids leads to -ketoglutarate and oxaloacetate.

43. Each NADH yields ≈ 3ATP Each FADH2 yields ≈ 2ATP Total yields ≈ 38ATP for each fully oxidized glucose.

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45. 2Ac-CoA + 2NAD+ + FAD OAA + 2CoA + 2NADH +FADH2 + 2H+ Glyoxylate cycle • The glyoxylate cycle results in the net conversion of two acetyl-CoA to succinate instead of 4 CO2 in citric acid cycle. • Succinate is transferred to mitochondrion where it can be converted to OAA (TCA) • Can go to cytosol where it is converted to oxaloacetate for gluconeogenesis. Net reaction Plants are able to convert fatty acids to glucose through this pathway

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