Ch 16. Citric Acid Cycle

1. Ch 16. Citric Acid Cycle • Pyruvate is converted to acetyl-CoA and CO2 by pyruvate dehydrogenase complex (E1,E2, E3) One NADH is generated, (NADH => 3 ATP in ox-phos) • Acetyl-CoA + oxaloacetate –> citrate • In 7 reactions 2 carbons are converted to CO2 and oxaloacetate is regenerated Products: 3 NADH (x 3) + FADH2 (x 2) + GTP = 12 ATP • Intermediates are substrates for biosynthesis • Reactions take place in mitochondria

6. Generation of Acetyl-CoA • Coenzyme A receives 2 carbons from pyruvate • Thioester linkage • ∆G°’=-31kJ mol-1 • Multi-enzyme process • Pyruvate + CoA + NAD+ –> acetyl CoA + CO2 + NADH

7. Pyruvate dehydrogenase multienzyme complex • Pyruvate dehydrogenase - E1 • Pyruvate + TPP –> Hydroxyethyl TPP + CO2 • Dihydrolipoyl transacetyase - E2 • HOEt-TPP + lipoamide–> Acetyl-dihydrolipoamide + TPP • Acetyl-dihydrolipoamide + CoA-SH –> acetyl CoA + dihydrolipoamide • Dihydrolipoyl dehydrogenase - E3 • NAD+ + dihydrolipoamide –> NADH + H+ + lipoamide

8. Pyruvate dehydrogenase • Thiamine Pyrophosphate (TPP) • Attacks carbonyl, releases CO2 • Hydroxyethyl TPP remains bound

9. Lipoamide flexible redox arm • Covalent E2 cofactor • S-S can be reduced to SH, SH • Lipoamide is a target for arsenic toxicity

10. Dihydrolipoyl transacetyase • Lipolysyl side chain extends to E1 • Transfers hydroxyethyl from TPP to Dihydrolipoamide • Transfers Acetyl group to CoA

11. Dihydrolipoyl dehydrogenase • Exchange of enzyme disulfide for lipoamide • E3 S-S + dihydrolipoamide –> E3–SH, SH + lipoamide • Bound FAD cofactor oxidizes E3 to disulfide • E3– SH, SH + FAD –> E3 S-S + • NAD+ oxidizes FADH2 to regenerate FAD • E3FADH2 + NAD+ –> E3FAD + NADH+ + H+

12. Control of pyruvate dehydrogenase • Product Inhibition by NADH and acetyl-CoA • Competitive inhibition of substrate binding • E2 and E3 are reversible • Phosphorylation inactivates mammalian E1 • Pyruvate dehydrogenase kinase • Stimulated by NADH and acetyl-CoA • Pyruvate dehydrogenase phosphatase - activates E1 • Activated by Ca2+ as part of Insulin response • Provides Acetyl CoA for fatty acid synthesis

13. Citric Acid Cycle worksheet

14. Acetyl-CoA Citric Acid Cycle intermediates Pyruvate Oxaloacetate Citrate Malate Isocitrate Fumarate a-ketoglutarate Succinate Succinyl-CoA

15. Citric Acid Cycle products

16. Citric Acid Cycle Enzymes - I 1. Citrate synthase Oxaloacetate + Acetyl-CoA + H2O –> Citrate + CoASH 2. Aconitase Citrate –> Cis Aconitate –> Isocitrate 3. Isocitrate dehydrogenase Isocitrate + NAD+ <–>  ketoglutarate + CO2 + NADH + H+ 4. -Ketoglutarate Dehydrogenase  ketoglutarate + CoA-SH + NAD+ <–> Succinyl-CoA + CO2 + NADH + H+

17. Citric Acid Cycle Enzymes - II 5. Succinyl-CoA Synthetase Succinyl-CoA + GDP + Pi –> Succinate + GTP + CoA-SH 6. Succinate succinate Dehydrogenase Succinate + FAD –> FADH2 + Fumarate 7. Fumarase Fumarate + H2O –> Malate 8. Malate Dehydrogenase Malate + NAD –> Oxaloacetate + NADH + H+

18. COO- CH2 HO-C–COO- CH2 COO- Citrate synthase CoA S C=O H3C H2O COO- C=O CH2 COO- • Oxaloacetate binds - cleft closure • Acetyl-CoA binds to closed form only • Acid base catalysis forms Enolate of Acetyl-CoA • Nucleophilic attack on OAA C=O forms Citryl CoA • Citrate and CoA-SH are released CoASH

19. COO- CH2 HO-C–COO- CH2 COO- COO- CH2 C–COO- HO-CH COO- Aconitase COO- CH2 C–COO- CH2 COO- H2O • Transfers C3-OH of citrate to C2-OH of isocitrate • Cis Aconitate (C2-C3 double bond) intermediate • Iron sulfur cluster [4Fe - 4S] • Fluoroacetate is converted to Fluorocitrate inhibits aconitase H2O

20. COO- CH2 C–COO- HO-CH COO- COO- CH2 C–COO- C=O COO- COO- CH2 CH2 C=O COO- Isocitrate dehydrogenase • Isocitrate + NAD+ <–>  ketoglutarate + CO2 + NADH + H+ • Initial oxidation of isocitrate to oxalosuccinate intermediate? • Decarboxylation to Give CO2 and  ketoglutarate CO2

21. COO- CH2 CH2 C=O COO- COO- CH2 CH2 C=O S–CoA -Ketoglutarate Dehydrogenase CO2 NADH H+ CoASH NAD • Similar to Pyruvate Dehydrogenase • Product is Succinyl CoA instead of Acetyl CoA • Multienzyme complex • E1- dehydrogenase, E2- dihydrolipoyl transsuccinylase, E3 identical to PD-E3

22. COO- CH2 CH2 C=O S–CoA Succinyl-CoA Synthetase COO- CH2 CH2 COO- GTP CoASH GDP Pi • Products Succinate, GTP and CoASH • ATP instead of GTP in plants and bacteria • Thioester free energy almost entirely conserved in phospho anhydride • Phosphoryl enzyme intermediate

23. Succinate Dehydrogenase H COO- C C -OOC H COO- CH2 CH2 COO- • Electrons accepted by covalently bound FAD • Embedded in mitochondrial membrane • FADH2 reoxidized by electron transport chain E-FADH2 E-FAD

24. Fumarase H COO- C C -OOC H COO- HO–CH CH2 COO- • H2O added across fumarate double bond to give Malate H2O

25. Malate Dehydrogenase COO- HO–CH CH2 COO- COO- C=O CH2 COO- • Hydride transfer from malate to NAD regenerates oxaloacetate for another cycle • Reaction pulled by citrate synthase consumption of OAA NADH H+ NAD+

26. Citric Acid Cycle

27. Citric Acid Cycle Regulation • Rate limiting steps (negative DG°’) • Citrate Synthase • Inhibited by citrate, NADH and Succinyl CoA • Isocitrate Dehydrogenase • Inhibited by ATP, NADH • Activated by Ca2+ and ADP • a-Ketoglutarate Dehydrogenase • Inhibited by NADH and Succinyl CoA • Activated by Ca2+

29. Anabolic uses of cycle intermediates • Gluconeogenesis (Ch 14) • Malate –> OAA –> –> Glucose • Lipid Biosynthesis (Ch 21) • Citrate –> OAA + Acetyl CoA –> –> lipids • Amino Acid Biosynthesis (Ch 22) • OAA and a-ketoglutarate • Porphyrin Biosynthesis (Ch 22) • Succinyl CoA

31. Catabolic sources of cycle intermediates • Fatty acid oxidation • Succinyl CoA • Amino Acid degradation • Succinyl CoA • Transamination of Amino Acid s • OAA and a-ketoglutarate

32. The Glyoxylate Pathway • Converts acetyl-CoA to Glucose in plant glyoxosomes • Isocitrate Lyase bypasses decarboxylations in CAC • Isocitrate –> succinate + glyoxylate • Malate Synthase • Glyoxylate + Acetyl CoA –> Malate

33. The Glyoxylate Pathway