The Citric Acid a.k.a. Krebs Cycle

OUTLINE: Introduction and review Transport Glycogenolysis Glycolysis Introduction & overview; 2 phases Phase I Phase II Summary: logic, energetics, labeling studies Other sugars Pasteur: Anaerobic vs Aerobic Fermentations Lactate-lactate dehydrogenase Acetoacetate decarboxylase Ethanol-pyruvate decarboxylase & alcohol dehydrogenase Exam-1 material Pyruvate oxidation: aerobic fates of pyruvate Exam-2 material pyruvate dehydrogenase complex 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 Glycogen-o-lysis Glyco-lysis Pyruvate Oxidation Krebs' Cycle Phosphorylation Oxidative 1 The Citric Acid Cycle a.k.a. Krebs Cycle, a.k.a. Tricarboxylic Acid Cycle (TCA) Time B.C. (Before the Cycle) Otto Warburg 1883-1970 Warburg Apparatus Manometer Substrates -respiration (e.g., glucose) -Measure rates of O2 consumption Tissues CO2 trap CO2 à H2CO3 à 2H+ + CO3-2 à K2CO3 + H2O 2 Time B.C. (Before the Cycle) O-leic M-alonic S-uccinic G-lutaric A-dipic P-imelic In 1920 BC, what was known about respiration? 1) Glycolysis gives rise to pyruvate 2) Adding 1 mole pyruvate to respiring tissues in a Warburg apparatus, there are 2.5 moles O2 1/2O 221/2 à2à à 23CO O22++CC33HH44OO33à àà 3CO + 2H O 2 + 2H2O consumed: 3) Any intermediate in the process will be oxidized at a rate ≥pyruvate 4) Many intermediates were tried, but few met this criteria, they were: succinate, fumarate, malate, alpha-ketoglutarate, etc. Albert Szent-Györgyi a-ketoglutarate glutarate à succinate à fumarate à malate à oxaloacetate | citrate à aconitate à isocitrate 6) In 1937, with help of German biochemist Franz Koop, Carl Martinus, demonstrated a series of reactions using citrate that produced aketoglutarate. Thus tricarboxylic acid and dicarboxylic acids would be interconverted with loss of CO2, but also support respiration. 1893-1986 5) Others had already worked out several compounds and their interconversion. Specifically, Albert Szent-Györgi had worked out the interconversion of the dicarboxylic acids. Carl Martinus worked out the interconversion of the tricarboxylic acids 1/2 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 Hans Krebs 1900-1981 Krebs confirmed that the pathway was consistent with succinate, fumarate, and malate. And these 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 3 Time B.C. (Before the Cycle) O-leic M-alonic S-uccinic G-lutaric A-dipic P-imelic 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. 4 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. 5 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 6 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 7 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 Realize that biochemists could distinguish the two acetates by running the citrate synthase reaction with H2 18 O. (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 ⊕ ⊕ 8 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+ 9 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. 10 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 11