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 H 2 O  G o’ = -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 CO 2

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 CO 2 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  FADH 2 –Lipollysyl ox’d back to ring w/ disulfide FADH 2 regen’d by NAD+ entry –FADH 2 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 CO 2 (not same 2 C’s that entered) 4 redox rxn’s –3 NAD+  3 NADH; 1 FAD  FADH 2 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 CH 3 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 H 2 O Cis-aconitate intermediate Iron-sulfur center Prod rapidly consumed in next step

33. Isocitrate   Ketoglutarate + CO 2

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.  Ketoglutarate  SuccinylCoA + CO 2

36.  Ketoglutarate Dehydrogenase Complex Identical rxn to PDC Similar E1, E2, E3 enzymes –E1 aa’s differ, bind  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 PO 4 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 +  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

52. Balance of amphibolic pathways through anapleurotic rxns –Replenish cycle intermediates so TCA remains constant –4 impt rxns –Synth oxaloacetate or malate from pyruvate or phosphoenolpyruvate Where did you see these reactants?

55. –If  glycolysis (so  PEP/pyr products), but not enough oxaloacetate to fuel cycle Cell can use excess PEP/pyr to make more oxaloacetate Now have sufficient to react w/ excess acetylCoA (from excess pyr, from excess PEP)