1. Citric Acid Cycle What is it? Series of rxns that oxidize acetyl CoA to 2CO 2 in a manner that conserves the liberated free energy for ATP production Breakdown products of glc, fats and amino acids are all oxidized via the cycle Intermediates also a source for many biosynthetic pathways - “amphibolic” which means serves in both catabolic and anabolic processes Also called “TCA cycle” - tricarboxylic acid cycle or “Krebs cycle”

2. Citric Acid Cycle Cellular respiration

3. Citric Acid Cycle Pyruvate dehydrogenase complex 3 mitochondrial euk enzymes 5 coenzymes

4. Citric Acid Cycle Pyruvate dehydrogenase complex 5 coenzymes - Coenzyme A (pantothenate) Reactive thiol is an acyl carrier

5. Citric Acid Cycle Pyruvate dehydrogenase complex 5 coenzymes - NAD + (niacin) Involved in redox, electron carrier

6. Citric Acid Cycle Pyruvate dehydrogenase complex 5 coenzymes - FAD (riboflavin, vitamin B2) Involved in redox, electron carrier

7. Citric Acid Cycle Pyruvate dehydrogenase complex 5 coenzymes - TPP (thiamine, vitamin B1) Helps in cleavage of bonds next to carbonyl

8. Citric Acid Cycle Pyruvate dehydrogenase complex 5 coenzymes - lipoate Reversible oxidation at thiols, serves as electron carrier and acyl carrier

9. Citric Acid Cycle Pyruvate dehydrogenase complex

10. Citric Acid Cycle Pyruvate dehydrogenase complex - regulation Eukaryotic complex regulated by phosphorylation Regulation Pyruvate: substrate for complex ADP: indicates low energy charge NADH, acetyl CoA: endproducts of complex Ca 2+ : certain hormones (insulin) raise [Ca 2+ ]

11. Citric Acid Cycle Every turn of cycle: 1. Joining an acetyl group to oxaloacetate 2. Oxidizing 2 carbons off as CO 2, leaving succinate 3. Converting succinate back to oxaloacetate 4. Producing 1 GTP and reduced cofactors

12. Citric Acid Cycle Net reaction: AcetylCoA + 3NAD + + FAD + GDP + P i + 2H 2 O  2CO 2 + CoA + 3NADH + FADH 2 + GTP + 2H +

13. Citric Acid Cycle Goal: capture energy from acetate  2CO 2 1. One GTP made: equivalent to 1 ATP 2. Lots of reduced cofactors, reducing power can be converted to ATP -  G irreversible  -ketoglutarate dehydrogenase complex -  G irreversible Only enzyme bound to inner mitochondrial membrane +  G made favorable by fast product removal

14. Citric Acid Cycle Regulation Rate of cycle is determined by: 1. Availability of substrates (acetyl CoA, NAD +, FAD) 2. Inhibition by accumulating products 3. Allosteric feedback inhibition of enzymes NAD + and FAD are regenerated by oxidative phosphorylation, which needs O 2

15. Citric Acid Cycle Regulation Three nonequilibrium rxns: 1. Citrate synthase 2. Isocitrate dehydrogenase 3.  -ketoglutarate dehydrogenase

16. Citric Acid Cycle Why so complicated? Hub of intermediary metabolism Greatest selective advantage

17. Citric Acid Cycle

18. Reaction 1 - Citrate synthase Condensation of acetylCoA with oxaloacetate to form citrate Irreversible

19. Citric Acid Cycle Reaction 2 - Aconitase Removal of H 2 O, stereospecifically add back H 2 O Reversible, pulled to right because isocitrate rapidly consumed Prochiral carbon allows for stereospecific rxn

20. Citric Acid Cycle Reaction 3 - isocitrate dehydrogenase Oxidative decarboxylation of isocitrate Irreversible

21. Citric Acid Cycle Reaction 4 -  -ketoglutarate dehydrogenase complex Oxidative decarboxylation of  -ketoglutarate Identical to pyruvate dehydrogenase reaction Three homologous enzymes (E1, E2, E3) Five coenzymes (TPP, lipoate, FAD, NAD +, CoA) Irreversible

22. Citric Acid Cycle Reaction 5 - Succinyl-CoA synthetase Hydrolysis of thioester bond energizes formation of GTP Synthetase indicates participation of nucleoside triphosphate Reversible

23. Citric Acid Cycle Reaction 6 - Succinate dehydrogenase Oxidation of succinate to fumarate Enzyme bound to inner mito membrane, only membrane-bound enz Reversible Malonate (analog of succinate) is a competitive inhibitor of enz, blocks activity of cycle

24. Citric Acid Cycle Reaction 7 - Fumarase Hydration of fumarate to L-malate Reversible Substrate Product

25. Citric Acid Cycle Reaction 8 - Malate dehydrogenase Oxidation of malate to oxaloacetate NAD + linked to enzyme High +  G˚, pulled to products by continual removal of oxaloacetate