Time B.C. (Before the Cycle)

Time B.C. (Before the Cycle) O-leic M-alonic S-uccinic G-lutaric glutarate à succinate à fumarate à malate à oxaloacetate A-dipic P-imelic citrate à aconitate à isocitrate “pyruvate” (acetate) Hans Krebs 1900-1981 Krebs confirmed that the pathway was consistent with succinate, fumarate, and malate proved to be useful because all these molecules increased oxygen consumption in the pigeon breast muscle. Dr. Kornberg: Lecture 02.08.17 (19:54-20:39) (1 min) The first clue came from an experiment with fumarate. Krebs did careful measurements using the Warburg manometer. Fumarate gave greater than expected oxygen consumption in the pigeon breast muscle. 3O2 + C4H4O4à4CO2 + 2H2O ∴1 µmole fumarate would consume 3 µmole O2 1)Malonic acid inhibition of the succinate à fumarate step prevented this increase…. BUT, succinate accumulated! 2)How can fumarate give rise to succinate? There must be a cycle 3)Tested by showing that using succinate or fumarate you could detect the formation of citrate. Later in 1937, he proposed that pyruvate would combine with oxaloacetate to make citrate in a cycle he called the Citric Acid Cycle. Later, Fritz Lipmann showed that it was acetyl-CoA and not pyruvate. 1 The Citric Acid Cycle The Citric Acid Cycle: Citrate Synthase • Joining of acetyl-CoA and oxaloacetate with C-C bond formation • Highly thermodynamically favorable/irreversible (DG°’ = –7.7 kcal/mol) – regulated by substrate availability and product inhibition – OH R2 – • Activity largely depends on [oxaloacetate]. • Rate-limiting step of CAC • Uses acid/base catalysis R2 – Carbonyl of oxaloacetate is a good electrophile. – Methyl of acetyl-CoA is not a good nucleophile… – …unless activated by deprotonation to form a carbanion. 2 The Citric Acid Cycle: Citrate Synthase Mechanism • Conformational change occurs upon binding oxaloacetate. • Avoids unnecessary hydrolysis of thioester in acetyl-CoA a) Open conformation: Free enzyme does not have a binding site for acetyl-CoA. Ordered binding. Sequential ordered ter bi C (H2O) b) Closed conformation: Binding of OAA creates binding for acetylCoA. Reactive carbanion is protected. The Citric Acid Cycle: Citrate Synthase Mechanism carbanion 3 The Citric Acid Cycle: Citrate Synthase Mechanism Hydrolysis of Thioester; citroyl-CoA 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 4 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 ⊕ ⊕ 5 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+ 6 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 O H 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 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 Isocitrate – Ferrous Wheel H: – OH --- O 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. 7 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) 8 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 (C-C bond b-to carbonyl). • This requires a 2-step process: –First perform an alcohol-to-keto dehydrogenation at C2 using NAD+ –Second, using carbonyl, 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 Vertebrate Aldolase (protonation of the carbanion) This mechanism is just like The Mn++ cofactor stabilizes the athat of LDH or other keto acid, which destabilizes the dehydrogenases (Base middle carboxylate (electron sink) 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–) 9 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 (TPP, lipoate, FAD) 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 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 10 The Citric Acid Cycle: The a-KetoGlutarate Dehydrogenase Complex – CH OOC–CH2– – CO O –OOC 2 2 CH2 2 Succinyl-CoA 2 a-ketoglutarate Succinyl 2 CH2COO Hydroxybutyryl S S – HS HS 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) 11