Understanding the Citric Acid Cycle: A Detailed Guide

OUTLINE: Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic Fermentations Pyruvate pyruvate dehydrogenase Krebs’ Cycle How did he figure it out? Overview 8 Steps Citrate Synthase Aconitase Isocitrate dehydrogenase Ketoglutarate dehydrogenase Succinyl-CoA synthetase Succinate dehydrogenase Fumarase Malate dehydrogenase The Citric Acid Cycle Citrate Synthase ✓ glutarate a-ketoglutarate à succinate à fumarate à malate à oxaloacetate | citrate à aconitate à isocitrate “pyruvate” (acetate) O= H3 C–C–SCoA 1 The Citric Acid Cycle: Aconitase Isomerization by Dehydration/Rehydration aconitase •Elimination of H2O from the symmetrical molecule, citrate, gives a cis C=C bond. aconitase – lyase •Rationale: – Citrate, a tertiary alcohol, is a poor substrate for oxidation. – Isocitrate, a secondary alcohol, is a good substrate for oxidation. •Thermodynamically unfavorable/reversible (DG°’ = +3.2 kcal/mol) – product concentration kept low to pull forward; citrate tends to “pool” with higher conc. • Dehydration & Addition of H2O to cis-aconitate is stereospecific. – This was initially very confusing to bio/organic chemists – Only R-isocitrate is produced by aconitase. – A biochemist names A.G. Ogston clarified the situation by realizing that the enzyme spatially templates this symmetrical molecule by binding in only one way (e.g., clockwise or counter clockwise, not both) – Distinguished by three-point attachment to the active site The Citric Acid Cycle: Aconitase 3-point attachment; prochirality This binding protects the nascent acetate from chemistry CH2COO– C Enzyme surface ⊕ ⊕ OOC D-H D-H – CH2COO– HO Enzyme surface ⊕ ⊕ 2 The Citric Acid Cycle: Aconitase Iron-Sulfur Center in Aconitase The Citric Acid Cycle: Aconitase Iron-Sulfur Centers Most Iron-sulfur are involved in re-dox reactions (without proton transfers). In aconitase, the role is very different. Hydrogenase: H2 à 2H+ 3 The Citric Acid Cycle: Aconitase Water removal from citrate and subsequent addition to cis-aconitate are catalyzed by the iron-sulfur center: sensitive to oxidative stress. Mechanism cis-aconitate Isotope exchange studies have shown that the hydroxyl is lost to solvent (the same –OH removed is not held by the Fe-S cluster and added back). – This oxygen is lost as water, and the two cis-carboxylates SWITCH bringing the C2 close for attack sp2 carbon; attack of activated hydroxyl from solvent is liganded to Fe can only occur from one side, not the other. The Citric Acid Cycle: Aconitase Water removal from citrate and subsequent addition to cis-aconitate are catalyzed by the iron-sulfur center: sensitive to oxidative stress. Mechanism L-isocitrate cis-aconitate (R) –O Ferrous Wheel Fe O H – This oxygen is lost as water, and the two cis-carboxylates SWITCH bringing the C2 close for attack Isotope exchange studies have shown that the hydroxyl is lost to solvent (the same –OH removed is not held by the Fe-S cluster and added back). Isotope exchange studies have shown that the proton is the same and goes from C3 to C2. sp2 carbon; attack of activated hydroxyl from solvent is liganded to Fe can only occur from one side, not the other. 4 The Citric Acid Cycle: Aconitase Isomerization by Dehydration/Rehydration •Dehydration of H2O from the symmetrical molecule, citrate, gives a cis C=C bond. •Rehydration of H2O to cisaconitate is stereospecific. H The Citric Acid Cycle Citrate Synthase ✓ Aconitase ✓ glutarate a-ketoglutarate à succinate à fumarate à malate à oxaloacetate | citrate à aconitate à isocitrate “pyruvate” (acetate) 5 The Citric Acid Cycle: Isocitrate dehydrogenase isocitrate dehydrogenase • Converting the C2 hydroxyl to a keto destabilizes the C-C bond to the carboxylate at C3. • This requires a 2-step process: –First perform a alcohol-to-keto dehydrogenation at C2 using NAD+ –Second, allow for decarboxylation (the oxidation of the carboxylate to CO2, with the reduction of C3. –C2 is oxidized, C3 is reduced, Carboxylate is oxidized: Net oxidation is 2e– • Isozymes are specific for NADP+ (cytosolic) or NAD+ (mitochondrial). • Favorable but irreversible due to loss of CO2 (DG°’ = –2.0 kcal/mol) • Regulated by [ATP] (OMSGAP) The Citric Acid Cycle: Isocitrate dehydrogenase Mechanism (protonation of the carbanion) This mechanism is just like The Mn++ cofactor stabilizes the that of LDH or other a-keto acid, which destabilizes dehydrogenases (Base the middle carboxylate abstracts –OH proton, carbonyl forms, elimination This is an oxidative decarboxylation at carboxylate b-to carbonyl, so it does not need TPP!! Uses Mn & a-carbonyl of :H–) 6 The Citric Acid Cycle: Isocitrate dehydrogenase Mechanism (protonation of the carbanion) This mechanism is just like The Mn++ cofactor stabilizes the that of LDH or other a-keto acid, which destabilizes dehydrogenases (Base the middle carboxylate abstracts –OH proton, carbonyl forms, elimination This is an oxidative decarboxylation at carboxylate b-to carbonyl, so it does not need TPP!! Uses Mn & a-carbonyl of :H–) The Citric Acid Cycle: The a-KetoGlutarate Dehydrogenase Complex Oxidative Decarboxylation of an α-keto acid: α-Ketoglutarate Dehydrogenase 12; two at each face 24; two at each edge; 3 at each corner 8; one at each corner E196, E224, E324 • Last oxidative decarboxylation in TCA cycle – full oxidation of all carbons of glucose: • Takes two turns of the cycle • The carbons oxidized are not directly from glucose because the carbons came from oxaloacetate, not acetyl-CoA • Requires TPP, FAD, Lipoic acid cofactors • Succinyl-CoA is another higher-energy thioester bond. • Highly thermodynamically favorable/irreversible Where have we seen this before? (DG°’ = –8.0 kcal/mol) – regulated by product inhibition 7 The Citric Acid Cycle: The a-KetoGlutarate Dehydrogenase Complex (a-KGDH) TPP Lipoic acid FAD TPP Lipoic acid FAD • Complex similar to pyruvate dehydrogenase – same coenzymes, identical mechanisms, E2 & E3 are identical – active site of E1 different to accommodate different-sized substrates The Citric Acid Cycle: The a-KetoGlutarate Dehydrogenase Complex (a-KGDH) TPP Lipoic acid FAD TPP Lipoic acid FAD • Complex similar to pyruvate dehydrogenase – same coenzymes, identical mechanisms, E2 & E3 are identical – active site of E1 different to accommodate different-sized substrates 8 The Citric Acid Cycle: The a-KetoGlutarate Dehydrogenase Complex – CH OOC–CH2– – CO 2 O –OOC 2 CH2 2 Succinyl-CoA 2 a-ketoglutarate Succinyl 2 CH2COO Hydroxybutyryl – a-ketoglutarate The Citric Acid Cycle Citrate Synthase ✓ ✓ ✓ Aconitase Isocitrate dehydrogenase a-ketoglutarate dehydrogenase complex glutarate a-ketoglutarate à succinate à fumarate à malate à oxaloacetate | citrate à aconitate à isocitrate “pyruvate” (acetate) 9