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2. The Citric Acid Cycle (CAC)

2. The Citric Acid Cycle (CAC). Pyruvate. CO 2. The sequence of events: Step 1: C-C bond formation to make citrate Step 2: Isomerization via dehydration/rehydration Steps 3–4: Oxidative decarboxylations to give 2 NADH Step 5: Substrate-level phosphorylation to give GTP

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2. The Citric Acid Cycle (CAC)

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  1. 2. The Citric Acid Cycle (CAC) Pyruvate CO2

  2. The sequence of events: • Step 1: C-C bond formation to make citrate • Step 2: Isomerization via dehydration/rehydration • Steps 3–4: Oxidative decarboxylations to give 2 NADH • Step 5: Substrate-level phosphorylation to give GTP • Step 6: Dehydrogenation to give reduced FADH2 • Step 7: Hydration • Step 8: Dehydrogenation to give NADH 2. The Citric Acid Cycle (CAC)

  3. List of enzymes involved: • Synthase • Catalyzes a synthesis process • Aconitase • A stereo-specific isomerization • Dehydrogenase • Removes hydrogen as H2 • Synthetase • Links two molecules by using the energy of cleavage of a pyrophosphate group • Fumarase • Catalyzes reversible hydration/rehydration of fumarate to malate 2. The Citric Acid Cycle (CAC)

  4. Step 1: C-C Bond Formation by Condensation of Acetyl-CoA and Oxaloacetate

  5. Citrate Synthase Reaction- • Condensation of acetyl-CoA and oxaloacetate • The only reaction with C-C bond formation • Rate-limiting step of CAC Mechanism- • Uses Acid/Base Catalysis • Carbonyl of oxaloacetate is a good electrophile • Methyl of acetyl-CoA is NOT a good nucleophile but isactivated by deprotonation Highly thermodynamically favorable/irreversible • Regulated by substrate availability and product inhibition • Activity largely depends on [oxaloacetate]

  6. Induced Fit in the Citrate Synthase Citrate Synthase has two subunits that create two binding sites for binding both oxaloacetate and acetyl-CoA. Binding is very conformation dependent: A. Open conformation • Free enzyme does not have a binding site for acetyl-CoA B. Closed conformation • Binding of OAA enables binding for acetyl-CoA • The conformation avoids hydrolysis of thioester in acetyl-CoA • Protects reactive carbanion

  7. Induced Fit in the Citrate Synthase

  8. Mechanism of Citrate Synthase: Acid/Base Catalysis

  9. Mechanism of Citrate Synthase: Acid/Base Catalysis

  10. Mechanism of Citrate Synthase: Hydrolysis of Thioester

  11. Step 2: Isomerization by Dehydration/ Rehydration

  12. Aconitase Key points: • Elimination of H2O from citrate gives a cis C=C bond • Lyase • Citrate, a tertiary alcohol, is a poor substrate for oxidation • Isocitrate, a secondary alcohol, is a good substrate for oxidation • Addition of H2O to cis-aconitate is stereospecific • Thermodynamically unfavorable/reversible • Product concentration kept low to pull forward

  13. Iron-Sulfur Center in Aconitase Water removal from citrate and subsequent addition to cis-aconitate are catalyzed by the iron-sulfur center: sensitive to oxidative stress.

  14. Aconitase is stereospecific Only R-isocitrate is produced by aconitase Distinguished by three-point attachment to the active site

  15. Aconitase is stereospecific • Only R-isocitrate is produced by aconitase because citrate is prochiral with respect to binding to the active site. • -Distinguished by three-point attachment to the active site

  16. Step 3: Oxidative Decarboxylation #2

  17. Isocitrate Dehydrogenase Key points: • Oxidative decarboxylation • Lose a carbon as CO2 • Oxidation of the alcohol to a ketone • Transfers a hydride to NAD+ generating NADH • Cytosolic isozyme uses NADP+ as a cofactor • Highly thermodynamically favorable/irreversible • Regulated by product inhibition and ATP

  18. Mechanisms of Isocitrate Dehydrogenase: Metal Ion Catalysis (Oxidation) 0 +2

  19. Mechanisms of Isocitrate Dehydrogenase: Metal Ion Catalysis (Decarboxylation) Carbon lost as CO2 did NOT come from acetyl-CoA.

  20. Mechanisms of Isocitrate Dehydrogenase: Rearrangement and Product Release

  21. Step 4: Final Oxidative Decarboxylation

  22. -Ketoglutarate Dehydrogenase Key points: • Last oxidative decarboxylation • Net full oxidation of all carbons of glucose • Carbons not directly from glucose because carbons lost came from oxaloacetate • Succinyl-CoA is another higher-energy thioester bond • Highly thermodynamically favorable/irreversible • Regulated by product inhibition

  23. -Ketoglutarate Dehydrogenase • Complex similar to pyruvate dehydrogenase • Same coenzymes, identical mechanisms • Active sites different to accommodate different-sized substrates

  24. Origin of C-atoms in CO2 • Both CO2 carbon atoms derived from oxaloacetate. • At this point in the metabolic pathway, a total of 6 CO2 are produced.

  25. Step 5: Generation of GTP through Thioester

  26. Succinyl-CoA Synthetase Key points: • Substrate level phosphorylation • Energy of thioester allows for incorporation of inorganic phosphate • Goes through a phospho-enzyme intermediate • Produces GTP, which can be converted to ATP • Slightly thermodynamically favorable/reversible • Product concentration kept low to pull forward

  27. Mechanism of Succinyl-CoA Synthetase

  28. GTP Converted to ATP • Catalyzed by nucleoside diphosphate kinase.

  29. Step 6:Oxidation of an Alkane to Alkene

  30. Succinate Dehydrogenase Key points: • Bound to mitochondrial inner membrane • Part of Complex II in the electron-transport chain • Reduction of the alkane to alkene (reverse reaction) requires FADH2 • Reduction potential of NAD is too low • FAD is covalently bound, which is unusual • Near equilibrium/reversible • Product concentration kept low to pull forward

  31. Step 7: Hydration Across a Double Bond

  32. Fumarase Key points: • Stereospecific • Addition of water is always trans and forms L-malate • OH- adds to fumarate and then H+ adds to the carbanion • Cannot distinguish between inner carbons, so either can gain –OH • Slightly thermodynamically favorable/reversible • Product concentration kept low to pull reaction forward

  33. Stereospecificity of Fumarase

  34. Step 8: Oxidation of Alcohol to a Ketone

  35. Malate Dehydrogenase Key points: • Final step of the cycle • Regenerates oxaloacetate for citrate synthase • Highly thermodynamically UNfavorable/reversible • Oxaloacetate concentration kept VERY low by citrate synthase • Pulls the reaction forward

  36. 3. One Turn of the Citric Acid Cycle

  37. 3A. Net Result of the Citric Acid Cycle Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 H2O  2CO2+ 3NADH + FADH2 + GTP + CoA + 3H+ • Net oxidation of two carbons to CO2 • Equivalent to two carbons of acetyl-CoA • but NOT the exact same carbons • Energy captured by electron transfer to NADH and FADH2 • Generates 1 GTP, which can be converted to ATP

  38. 3B. Direct and Indirect ATP Yield

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