Fermentation - Acetoacetate Decarboxylase

OUTLINE: 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 Ethanol-pyruvate decarboxylase & alcohol dehydrogenase Acetoacetate decarboxylase Pyruvate pyruvate dehydrogenase Fermentation: Acetoacetate decarboxylase P P Strict anaerobe: Clostridium acetobutylicum In 1916, the enzyme, acetoacetate decarboxylase, was discovered by Chaim Weizmann….. on to serendipity! 1 Fates of Pyruvate No Oxygen (anaerobic) Pyruvate Oxidation Oxygen available (aerobic) Fates of Pyruvate No Oxygen (anaerobic) Pyruvate Oxidation Oxygen available (aerobic) Dr. Kornberg: Lecture 2 Pyruvate Oxidation ONLY when O2 is present • This is why its called cellular respiration. • The end result will be the COMPLETE oxidation of carbon to CO2 Pyruvate Oxidation Glycogen-o-lysis Glyco-lysis Pyruvate Oxidation Krebs' Cycle Phosphorylation Oxidative 3 Pyruvate Oxidation Glycolysis 2 GLUCOSE DG′° = –35 kcal/mol (–146 kJ/mol) Respiration (+ 6 O2) DG′° = –678 kcal/mol 6 CO2 + 6 H2O (–2,840 kJ/mol) • Process in which cells consume O2 and produce CO2 • Provides more energy (ATP) per glucose than glycolysis • Process is slower than glycolysis • Evolutionary origin: developed about 2,500 mya • Used by animals, plants, and many microorganisms • Other fuel sources (lipids and AA) converge on respiration. Occurs in three major stages: 1) Pyruvate oxidation (acetyl CoA production) 2) acetyl CoA oxidation (CO2 production) 3) electron transfer and oxidative phosphorylation (H2O production) Pyruvate Oxidation Localization: In Eukaryotes, Respiration is Localized to the Mitochondria (Compartmentation) • Glycolysis occurs in the cytoplasm. Mitochondria • Pyruvate Oxidation and Citric Acid Cycle occurs in the mitochondrial matrix.† • Fantastic example of Compartmentation. • Oxidative phosphorylation occurs in the inner membrane. Inner membrane Outer membrane Cristae Matrix †Except succinate dehydrogenase, which is located in the inner membrane 4 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. Pyruvate Oxidation PDC is a large (up to 10 MDa) multienzyme complex. -pyruvate dehydrogenase (E1) -dihydrolipoyl transacetylase (E2) -dihydrolipoyl dehydrogenase (E3) 500 Å PDC E2 complex Cryoelectronmicroscopy • 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. 5 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. 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 6 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 Pyruvate Oxidation Coenzyme A Enzyme 2 • Formation of acetyl-CoA: simple thio-ester exchange 7 Pyruvate Oxidation S S 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 Pyruvate Oxidation S S 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 8 Fermentation: Ethanol Humans are missing the gene for this enzyme. 9