1. BRIDGING REACTION STEP 2 Fall 2013 BIOT 309

2. TRANSITION OR BRIDGING REACTION Connects glycolysis to citric acid/Kreb’s Cycle OVERALL REACTION 2 pyruvate + 2 NAD + + 2 CoA-SH (coenzyme A) 2 acetyl-CoA + 2 NADH + 2 H + + 2 CO 2 CONNECTION TO OTHER BIOLOGY: Where else is CO 2 made?

3. TRANSITION REACTION 3 carbon 2 carbon Co A

4. STEP 3 AEROBIC RESPIRATION: Krebs Cycle BIOT 309 Fall 2013

5. Tricarboxylic Acid Cycle = Krebs Cycle = Citric acid Cycle

6. THE TCA CYCLE Converts acetyl CoA (from pyruvate via bridging reaction) to CO 2 Provides small amounts of energy in the form of GTP/ATP Collects electrons and stores as NADH and FADH 2  Electron Transport Chain (ETC) Provides intermediates for other pathways Occurs in cytoplasm

7. KREB’S CYCLE Summary Reaction: acetyl-CoA + 3NAD + + FAD + GDP + P i + 2H 2 O ——> 2CO 2 + HSCoA + 3NADH + FADH 2 + GTP + 2H +

8. TCA/KREB’S CYCLE

9. CITRIC ACID CYCLE

11. CHEMICAL REACTIONS Key Equation: Δ G 0 = -RTlnK eq Δ G 0 = Gibb’s standard free energy change, distance from equilibrium, (expresses driving force of reaction) K eq =[products]/[reactants]; measurable R= gas constant T = absolute temperature (Kelvin)

12. BIOCHEMICAL REACTIONS Instead of Δ G 0, Δ G 0’ is used Δ G 0’ = standard free energy change at pH 7.0 = biochemical standard free energy

13. Remember: enzymes, cofactors Lower activation energy Accelerate reaction Organize and control reaction Recover energy in new chemical forms and make it available for other uses

14. Gibb’s Free Energy if Δ G 0’ is negative, reaction goes forward spontaneously; -  products have less energy than reactants if Δ G 0’ is ~ 0, reaction is at equilibrium if Δ G 0’ is positive, reaction does not go forward spontaneously Δ G 0’ of two or more reactions is calculated by adding reactions and the Δ G 0’ of the reactions CAVEAT: Δ G 0 values shown in next slides will not be true under all circumstances, could be different for prokaryotes and eukaryotes

15. KREB’S CYCLE, step 1 Citrate Synthase Aldol Condensation, X 2 C4 C6 C

16. KREB’S CYCLE, step 2 Aconitase Dehydration, Fe-S

17. KREB’S CYCLE, step 3 Aconitase Hydration, 4Fe-4S

18. Steps 2 & 3 combined STEPS 2 & 3 done by one enzyme aconitase Observe that: Step 2: dehydration generates (double bond) intermediate (cis-aconitate) Step 3: dehydration moves position of OH group

19. PRINCIPLE & EXAMPLE: Δ G 0’ of overall reaction is calculated by adding reactions and the Δ G 0’ of the reactions*: Applied to 2 or more reactions, e.g., all of EMP or TCA Δ G 0’ = +2 kcal/mol Δ G 0’ = -0.5 kcal/mol Δ G 0’ = +1.5 kcal/mol citrate  isocitrate citrate  cis-aconitate cis-aconitate  isocitrate

20. KREB’S CYCLE, step 4 Isocitrate Dehydrogenase 2 step reaction Oxidative decarboxylation, Mg 2+ or Mn 2+ NAD + NADH, H + 6 C5 C SPONTANEOUS

21. KREB’S CYCLE, step 5 α-Ketoglutarate Dehydrogenase Complex Oxidative Decarboxylation, TPP, Lipoic Acid, FAD NAD + + CoA-SH NADH, H + 5 C4 C SPONTANEOUS

22. KREB’S CYCLE, step 6 Succinyl CoA Synthetase Substrate Level Phosphorylation, FAD, TPP, Lipoic Acid GTP converted into ATP by nucleoside diphosphate kinase

23. KREB’S CYCLE, step 7 Succinate Dehydrogenase Oxidation, FAD & FeS Why FAD? alkane oxidation poorly exergonic and can’t reduce NAD +

24. KREB’S CYCLE, step 8 Fumarate Hydratase Hydration, Fe-S

25. KREB’S CYCLE, step 9 Malate Dehydrogenase Oxidation Δ G 0’ = +7 kcal/mol

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28. KREB’S CYCLE !!! Summary Reaction: acetyl-CoA + 3NAD + + FAD + GDP + P i + 2H 2 O ——> 2CO 2 + HSCoA + 3NADH + FADH 2 + GTP + 2H +

29. Transition Reaction + Kreb’s Cycle Summary Reaction: 1 pyruvate + 4 NAD + + 1 FAD + 1 GDP + 1 Pi ——> 4 CO 2 + 4 NADH + 4 H + + 1 FADH 2 + 1 GTP(1 ATP)

30. EMP + TR + TCA Summary Reaction: GLUCOSE + 2H 2 0 + 10 NAD+ 2 FAD + 4 ADP + 4 Pi ——> 6 CO 2 + 10 NADH + 10 H + + 4 ATP + 2FADH 2

31. GLYOXYLATE CYCLE KREBS CYCLE ALTERNATIVE BIOT 309 Fall 2013

32. GLYOXYLATE SHUNT/CYCLE By-passes 2 decarboxylation steps in TCA making possible – net formation of succinate, oxaloacetate, and other cycle intermediates from acetyl-CoA Retains the two carbons lost in decarboxylation steps with each turn of TCA => net synthesis of oxaloacetate, a four-carbon molecule, because each turn of the cycle incorporates two molecules of acetyl-CoA – Oxaloacetate used for other purposes

33. GLYOXYLATE SHUNT/CYCLE Allows many bacteria to metabolize two- carbon substrates such as acetate FOR EXAMPLE: E. coli can be grown in a medium that provides acetate as the sole carbon source. E. coli synthesize acetyl-CoA, then uses it for energy production (via the citric acid cycle)

34. GLYOXYLATE SHUNT/CYCLE Some enzymes in common with TCA BUT has two exclusive enzymes not in TCA – isocitrate lyase: cleaves D-isocitrate to glyoxylate and succinate – malate synthase: forms L-malate from glyoxylate and acetyl-CoA

35. GLYOXYLATE SHUNT/CYCLE Used when the principal or sole carbon source is a C2 compound (acetate, ethanol). Fat catabolism produces acetyl CoA which feeds into other catabolic reactions and produces energy