Kreb’s Cycle

1. Kreb’s Cycle Chapter 16

2. Glycolysis: 6C Glu  3C Pyruvate x2 • Glu + 2NAD+ + 2 ADP + 2 Pi  2 pyr + 2 NADH + 2 H+ + 2 ATP + 2 H2O • D Go’ = -85 kJ/mole • 2 NADH  e- transport  ATP synth • In cytosol

3. 3C Pyruvate Product • 2 C’s added to Coenzyme A (CoA) • As acetate group • Activates CoA (thioester) • 1 C as CO2

6. Pyruvate Dehydrogenase Complex (PDC) • Catalyzes acetylation CoA • Oxidative decarboxylation (LEO + cleave carboxylate)

7. Pyruvate Dehydrogenase Complex (PDC) • In mitochondria • Sev copies of 3 associated enz’s • Pyruvate dehydrogenase (E1) • Dihydrolipoyl transacetylase (E2) • Dihydrolipoyl dehydrogenase (E3)

8. Book: mammalian PDC 5X size ribosome • Bovine: circular arrangement • 5 cofactors • Thiamine, riboflavin, niacin, pantothenate • Two regulatory proteins assoc’d • Kinase, phosphatase

11. PDC E1: Pyruvate Dehydrogenase • 24 copies in complex (E. coli) • Cofactor: thiamine pyrophosphate (TPP) • From Vitamin B1 (Chpt 14)

12. Pyr binds  ethanolic grp att’d to TPP • CO2 released • Ox’n to acetaldehyde att’d as hydroxyethyl • Acetaldehyde transferred to E2 of PDC (Chpt 14)

13. PDC E2: Dihydrolipoyl Transacetylase • “Core” of complex • 24 copies (E. coli); 60 copies (bovine) • Cofactor: lipollysyl • Molecular “arm” • In ox’d form – 5 membered ring w/ disulfide

15. Ethanolic grp to lipollysyl • Ox’d  acetaldehyde • -S-S- red’d to –SH HS- w/ ox’n to acetaldehyde • Forms thioester • Site of attack by CoASH • Transesterification •  AcetylCoA + dithiol lipoyl

17. PDC E3: Dihydrolipoyl Dehydrogenase • 12 copies att’d to E2 (E. coli) • Cofactor: FAD • REMEMBER: Flavin nucleotide cofactors bound to enz’s • (Nicotinamide nucleotides cofactors freer to dissociate) • Used to reoxidize lipollysyl

18. FAD red’d  FADH2 • Lipollysyl ox’d back to ring w/ disulfide • FADH2 regen’d by NAD+ entry • FADH2 ox’d  original FAD • NAD+ red’d  NADH • Leaves complex • Where might it go?

19. PDC Summary • 3 Enz’s closely assoc’d • Book: “substrate channeling” • Acetyl grp physically transferred • Regulatory • Both allosteric + covalently modified regulation • E1 has kinase, phosphatase enz’s assoc’d • Kinase phosphorylates, inactivates • Phosphatase dephosphorylates, activates

20. Assoc’d kinase allosterically controlled • ATP stimulates • Act’d kinase inactivates PDC • So  [ATP]  ?? PDC?? • Modulators • Inhibitory: ATP, NADH, acetyl CoA, fatty acids • Why?? • Stimulatory: ADP/AMP, NAD+, pyruvate, CoA • Why??

21. Kreb’s Cycle • = Citric Acid Cycle = Tricarboxylic Acid Cycle = TCA Cycle • 2 C’s from pyr (as acetyl on acetylCoA) • 2 C’s leave as CO2 (not same 2 C’s that entered) • 4 redox rxn’s • 3 NAD+  3 NADH; 1 FAD  FADH2 • Where will these go?

22. 1 high energy phosphate bond formed • 1 GDP  1 GTP (some cells 1 ADP  1 ATP) • REMEMBER the name of this phosph’n? • Oxaloacetate regen’d • REMEMBER: 2 turns for each glu • Up to 38 ATP/glu (>1160 kJ/mole avail) • 1 step uses complex sim to PDC

24. Acetyl CoA + Oxaloacetate  Citrate + CoASH

25. Citrate Synthetase • Condensation rxn • CoASH regen’d • Through CH3 of acetyl • Transient intermediate: citroyl CoA • Energy rel’d from cleavage acetylCoA • Why? What grps impt to exergonic rxn

26. Oxaloacetate binds first •  Conform’l change • Now site for acetylCoA

29. Modulators • Availability of substrates • Inhib’n w/  [citrate] • What type of inhib’n? •  [citrate] also inhibits PFK-1 • Where is PFK-1? • What type of inhib’n would this be? • Inhib’n w/  [ATP] • Relieved w/  [ADP] • Why? • Inhib’n w/  [succinyl CoA] • Feedback inhib’n

31. Citrate  Isocitrate

32. Aconitase • Isomerization • Through reversible add’n H2O • Cis-aconitate intermediate • Iron-sulfur center • Prod rapidly consumed in next step

33. Isocitrate a Ketoglutarate + CO2

34. Isocitrate Dehydrogenase • Ox’n rxn (oxidative decarboxylation) • Mn+2 coordinates/stabilizes intermediate • NAD+ or NADP+ depending on isozyme • Regulation • Inhib’n w/  [ATP] • Inhib’n w/  ratio [NADH]/[NAD+] • Why?

35. a Ketoglutarate  SuccinylCoA + CO2

36. a Ketoglutarate Dehydrogenase Complex • Identical rxn to PDC • Similar E1, E2, E3 enzymes • E1 aa’s differ, bind a ketoglutarate specifically • Same cofactors • Regulation • Inhib’n w/  [succinyl CoA] • Inhib’n w/  ratio [NADH]/[NAD+]

37. SuccinylCoA  Succinate + CoASH

38. SuccinylCoA Synthetase • Add’n Pi  high energy acyl phosphate intermediate in enz active site • CoASH released

39. Phosphate transferred to enz active site His • GDP enters active site; phosph’d  GTP • Substrate level phosph’n results • Book: GTP formed transfers PO4 to ADP later

40. Succinate  Fumarate

41. Succinate Dehydrogenase • Membr-bound • Euk’s – inner mitoch membr • Prok’s – plasma membr • Impt also in e- transport • Iron-sulfur centers + FAD • FAD may be cov’ly bound • Malonate is competitive inhibitor

42. Fumarate  L-Malate

43. Fumarase • Hydration trans across db • Enz stereo-specific

44. L-Malate  Oxaloacetate

45. L-Malate Dehydrogenase • Substrate limited rxn • Large + D G • Why does the rxn go?

47. Cycle • Complete w/ regen’n oxaloacetate • Regulation through • [substrate], [product] • Coenzymes • Nucleotide phosphates • Other nutrient pathways

49. Catabolism/Anabolism Balanced through Kreb’s Cycle • Amphibolic • Impt to both catabolism (breakdown) and anabolism (build-up) of cell’s molecules • Catabolism of carbohydrates, FA’s, aa’s through pyruvate, acetylCoA Kreb’s  ATP • Anabolism by cycle intermediates  aa’s, fa’s, lipids, purines/pyrimidines