1. CHAPTER 16 The Citric Acid Cycle –Cellular respiration –Conversion of pyruvate to activated acetate –Reactions of the citric acid cycle –Regulation of the citric acid cycle –Conversion of acetate to carbohydrate precursors in the glyoxylate cycle Key topics:

2. Only a Small Amount of Energy Available in Glucose is Captured in Glycolysis 2  G’° = -146 kJ/mol Glycolysis Full oxidation (+ 6 O 2 )  G’° = -2,840 kJ/mol 6 CO 2 + 6 H 2 O GLUCOSE

3. Cellular Respiration process in which cells consume O 2 and produce CO 2 provides more energy (ATP) from glucose than glycolysis also captures energy stored in lipids and amino acids evolutionary origin: developed about 2.5 billion years ago used by animals, plants, and many microorganisms occurs in three major stages: - acetyl CoA production - acetyl CoA oxidation - electron transfer and oxidative phosphorylation

4. Respiration: Stage 1 Generates some: ATP, NADH, FADH 2

6. Respiration: Stage 2 Generates more NADH, FADH 2 and one GTP

8. Respiration: Stage 3 Makes lots of ATP

10. In Eukaryotes, Citric Acid Cycle Occurs in Mitochondria Glycolysis occurs in the cytoplasm Citric acid cycle occurs in the mitochondrial matrix † Oxidative phosphorylation occurs in the inner membrane † Except succinate dehydrogenase, which is located in the inner membrane

12. Conversion of Pyruvate to Acetyl-CoA net reaction: oxidative decarboxylation of pyruvate acetyl-CoA can enter the citric acid cycle and yield energy acetyl-CoA can be used to synthesize storage lipids requires five coenzymes catalyzed by the pyruvate decarboxylase complex

14. Pyruvate Dehydrogenase Complex (PDC) PDC is a large (M r = 7.8 × 10 6 Da) multienzyme complex - pyruvate dehydrogenase (E 1 ) - dihydrolipoyl transacetylase (E 2 ) - dihydrolipoyl dehydrogenase (E 3 ) short distance between catalytic sites allows channeling of substrates from one catalytic site to another channeling minimizes side reactions activity of the complex is subject to regulation (ATP)

15. Cryoelectronmicroscopy of PDC Samples are in near-native frozen hydrated state Low temperature protects biological specimens against radiation damage Electrons have smaller de Broglie wavelength and produce much higher resolution images than light

17. Three-dimensional Reconstruction from Cryo-EM data

19. Sequence of Events in Pyruvate Decarboxylation Step 1: Decarboxylation of pyruvate to an aldehyde Step 2: Oxidation of aldehyde to a carboxylic acid Step 3: Formation of acetyl CoA Step 4: Reoxidation of the lipoamide cofactor Step 5: Regeneration of the oxidized FAD cofactor

21. Chemistry of Oxidative Decarboxylation of Pyruvate NAD + and CoA-SH are co-substrates TPP, lipoyllysine and FAD are prosthetic groups

23. Structure of CoA Recall that coenzymes or co-substrates are not a permanent part of the enzymes’ structure; they associate, fulfill a function, and dissociate The function of CoA is to accept and carry acetyl groups

25. Structure of Lipoyllysine Recall that prosthetic groups are strongly bound to the protein. In this case, the lipoic acid is covalently linked to the enzyme via a lysine residue.

27. The Citric Acid Cycle

29. Sequence of Events in the Citric Acid Cycle Step 1: C-C bond formation to make citrate Step 2: Isomerization via dehydration, followed by hydration Steps 3-4: Oxidative decarboxylations to give 2 NADH Step 5: Substrate-level phosphorylation to give GTP Step 6: Dehydrogenation to give reduced FADH 2 Step 7: Hydration Step 8: Dehydrogenation to give NADH

31. Net Effect of the Citric Acid Cycle Acetyl-CoA + 3NAD + + FAD + GDP + P i + 2 H 2 O 2CO 2 +3NADH + FADH 2 + GTP + CoA + 3H + carbons of acetyl groups in acetyl-CoA are oxidized to CO 2 electrons from this process reduce NAD + and FAD one GTP is formed per cycle, this can be converted to ATP intermediates in the cycle are not depleted

32. Direct and Indirect ATP Yield

34. Role of the Citric Acid Cycle in Anabolism

36. Regulation of the Citric Acid Cycle