10 TCA Cycle V2026

BB 422/622 phospho-fructo-kinase (PFK-1)Aldolase- Schiff base (electron sink to stabilize a carbanion) triose-phosphate isomerase (TPI)- endiol OUTLINE: Review of 421 Goals of 422 Review of chemical principles Thermodynamics C/O cycles Overview of Metabolism ATP cycles Energy Coupling Chemical Reactivity Bioenergetics Membrane Transport Review of membrane structure, dynamics, and proteins Mediated/non-mediated Energetics Facilitative Diffusion ionophores GLUT1 Aquaporins Potassium channel Active Transport Primary Na/K pump ABC transporters Secondary Glc import Bicarbonate/Cl Lactose/H+ Catabolism of Glucose Glycogenolysis phosphorylase debranching enzyme phospho-gluco-mutase (PGM) Glycolysis Introduction & overview; Phase I hexokinase- phosphotransferase-coupling phospho-gluco-isomerase (PGI)- endiol Exam 1 Phase II GAPDH– oxidation PG kinase- return on investmentsubstrate-level phosphorylation PG mutase- acid/base; phospho-enzyme Enolase– enolate Pyruvate Kinase- phosphotransferase Summary: labeling studies, logic, energetics Catabolism of Other sugars Pasteur: Anaerobic vs Aerobic Fermentations- Anaerobic Lactate lactate dehydrogenase Acetoacetate decarboxylase Ethanol pyruvate decarboxylase alcohol dehydrogenase Aerobic Pyruvate pyruvate dehydrogenase complex Krebs’ Cycle How did he figure it out? Overview 8 Steps Citrate Synthase 8 Pyruvate Oxidation Conversion of Pyruvate to Acetyl-CoA • Net reaction: – oxidative decarboxylation of pyruvate • Means pyruvate will get oxidized as the carboxylate leaves (as CO2) – first carbons of glucose to be fully oxidized (C3 & C4) • Fairly simple reaction done by a complicated process. • Highly thermodynamically favorable/irreversible (DG°’ = –8 kcal/mol); mostly due to the loss of CO2 • Catalyzed by the Pyruvate Dehydrogenase Complex (PDC) – – – – – – Three main enzyme, each with multiple subunits: E1, E2, E3 Regulatory subunits: PD kinase & PD phosphatase Overall structure of E196, E224, E324 requires 5 coenzymes TPP, lipoic acid, and FAD are prosthetic groups. NAD+ and CoA-SH are co-substrates. 10 1 Pyruvate Oxidation PDC is a large (up to 10 MDa) multienzyme complex. -pyruvate dehydrogenase (E1) -dihydrolipoyl transacetylase (E2) -dihydrolipoyl dehydrogenase (E3) Cryo-Electron Microscopy 500 Å PDC E2 complex Nobel Prize for Chemistry in 2017 11 Pyruvate Oxidation PDC is a large (up to 10 MDa) multienzyme complex. -pyruvate dehydrogenase (E1) -dihydrolipoyl transacetylase (E2) -dihydrolipoyl dehydrogenase (E3) Cryoelectronmicroscopy : 500 Å PDC E2 complex • Samples are in a near-native frozen hydrated state. • Low temperature protects biological specimens against radiation damage. • Electrons have a smaller wavelength and produce much higher-resolution images than light. • No need for a crystal. 12 2 Pyruvate Oxidation Box in a box 12; two at each face E1 24; two at each edge; 3 at each corner 8; one at each corner int E196, E224, E324 E2 The lipoic acid is covalently linked to the enzyme via a lysine residue; becoming a Prosthetic group. E3 13 Pyruvate Oxidation Box in a box 12; two at each face E1 24; two at each edge; 3 at each corner 8; one at each corner int E196, E224, E324 E2 E3 The lipoic acid is covalently linked to the enzyme via a lysine residue; becoming a Prosthetic group. 14 3 Pyruvate Oxidation O CH 3 C E1 R–E2 H ❺ E2 R– :B–E2 E2–AH ❹ E2 R– Enzyme 1 • Steps 1-3: Decarboxylation of pyruvate to an enol • Step 4: Acylation of enol/carbanion to a thio-hemiacetal • Step 5: Electrons reduce lipoamide and form a thioester. Hydroxyethyl TPP 16 Pyruvate Oxidation Coenzyme A Enzyme 2 • Formation of acetyl-CoA: simple thio-ester exchange 17 4 Pyruvate Oxidation S S Why? HS HS Enzyme 3 • Step 4: Reoxidation of the lipoamide cofactor; reduction of FAD/Cys/Cys – – Disulfide interchange with dihydrolipoyllysine and 2 adjacent Cys in disulfide bond The reduced Cys reform disulfide using FAD, which funnels electrons to NAD+ • Step 5: Regeneration of the oxidized FAD/Cys/Cys active site – forming NADH 18 Pyruvate Oxidation *from Maeda-Yorita et al., (1994) Biochem. 33, 6213 E-FADH2 + NADH + H+ ßà NADH + H+ + E-FAD DE°’ = E°’ (reduction) – E°’ (oxidation) DE°’ = E°’ (NAD+) – E°’ (FAD) Thorpe, C. & Williams, C. H. Jr. (1981). Lipoamide dehydrogenas nucleotide induced changes in monoalkylated two-electron reduced 1507-1513. Mattevi, A., Schierbeek, A. J. & Hol, W. G. (1991). Refined crystal dehydrogenase from Azotobacter vinelandii at 2.2 A resolution. J. M = –0.320 V – (+0.031 V*) DG°’ = –0.351 V = –n F DE°’ = –(2)(23.06kcalV-1mol-1)(–0.351 V) = +16 kcal mol-1 21 5 Pyruvate Oxidation *from Maeda-Yorita et al., (1994) Biochem. 33, 6213 E-FADH2 + NADH + H+ ßà NADH + H+ + E-FAD DE°’ = E°’ (reduction) – E°’ (oxidation) DE°’ = E°’ (NAD+) – E°’ (FAD) Thorpe, C. & Williams, C. H. Jr. (1981). Lipoamide dehydrogenas nucleotide induced changes in monoalkylated two-electron reduced 1507-1513. Mattevi, A., Schierbeek, A. J. & Hol, W. G. (1991). Refined crystal dehydrogenase from Azotobacter vinelandii at 2.2 A resolution. J. M = –0.320 V – (+0.031 V*) = –0.351 V DG°’ = –n F DE°’ = –(2)(23.06kcalV-1mol-1)(–0.351 V) = +16 kcal mol-1 22 Pyruvate Oxidation SUMMARY Enzyme 1 • Step 1: Decarboxylation of pyruvate to an enol (hydroxyethyl-TPPl • Step 2: Acylation of enol to a thioester on lipoic acid. Enzyme 2 • Step 3: Formation of acetyl-CoA: simple thio-ester exchange Enzyme 3 • Step 4: Reoxidation of the lipoamide cofactor; reduction of FAD/Cys/Cys • Step 5: Regeneration of the oxidized FAD/Cys/Cys active site – forming NADH 23 6 Pyruvate Oxidation E196, E224, E324 E2 25 Pyruvate Oxidation Overall Reaction of PDC Pyruvate + Coenzyme-A (CoASH) + NAD+ ⇌ PDC (TPP, lipoic acid, FAD) DG°’ = –8 kcal/mol CO2 + Acetyl-Coenzyme-A (Ac-CoA) + NADH + H+ 26 7 Pyruvate Oxidation Overall Reaction of PDC PDC (TPP, lipoic acid, FAD) ⇌ Pyruvate + Coenzyme-A (CoASH) + NAD+ DG°’ = –8 kcal/mol CO2 + Acetyl-Coenzyme-A (Ac-CoA) + NADH + H+ Fates of Acetyl CoA Acetyl-Coenzyme-A Amino acids 1. Oxidation 2. Lipid metabolism 3. Amino acid metabolism 28 The Citric Acid Cycle Glycogen-o-lysis Glyco-lysis Pyruvate Oxidation Krebs' Cycle Phosphorylation Oxidative 29 8 The Citric Acid Cycle a.k.a. Krebs Cycle, a.k.a. Tricarboxylic Acid Cycle (TCA) 30 Time B.C. (Before the Cycle) Otto Warburg 1883-1970 Warburg Apparatus Manometer Substrates -respiration (e.g., glucose) -Measure rates of O2 consumption UTube instructions (http://youtu.be/M-HYbZwN43o) Tissues CO2 trap CO2 à H2CO3 à 2H+ + CO3-2 à K2CO3 + H2O KOH 31 9 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 pyruvate to respiring tissues in a Warburg apparatus, there are 2.5 O2 consumed: 1/2O 221/2 àà à 3CO + 2H O O ++CC HH OO à àà 3CO + 2H O 22 3) 4) 33 44 33 2 2 2 2 Any intermediate in the process will be oxidized at a rate ≥pyruvate 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 32 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 proved to be useful because all these molecules increased oxygen consumption in the pigeon breast muscle. 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 33 Dr. Kornberg: Lecture 02.08.17 (19:54-20:39) (1 min) 10 Time B.C. (Before the Cycle) 34 O-leic M-alonic S-uccinic G-lutaric A-dipic P-imelic Dr. Kornberg: Lecture 02.08.17 (19:54-20:39) (1 min) 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. He observed much more than 3:1!! The first clue came from an experiment with fumarate. Krebs did careful measurements using the Warburg manometer. Fumarate gave greater 3O2 + C4H4O4à4CO2 + 2H2O than expected oxygen consumption in the pigeon breast muscle. ∴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. 35 Dr. Kornberg: Lecture 02.08.17 (19:54-20:39) (1 min) 11 The Citric Acid Cycle • Step 1: C-C bond formation between acetate (2C) and oxaloacetate (4C) to make citrate (6C) • 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 FADH2 • Step 7: Hydration • Step 8: Dehydrogenation to give NADH 37 ⑧ ⑦ ⑥ ① ⑤ ② ④ ③ The Citric Acid Cycle: Citrate Synthase ter-bi reaction • 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. 38 12 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. 39 The Citric Acid Cycle: Citrate Synthase Mechanism carbanion • Resonance stabilized enolate/carbanion • Low barrier H-bond with His 274 41 13