TCA Cycle Pyruvate Oxidation

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: anaerobic fates of pyruvate Lactate-lactate dehydrogenase Acetoacetate decarboxylase Ethanol-pyruvate decarboxylase & alcohol dehydrogenase Pyruvate oxidation: aerobic fates of pyruvate pyruvate dehydrogenase complex Fates of Pyruvate No Oxygen (anaerobic) Pyruvate Oxidation Oxygen available (aerobic) Dr. Kornberg: Lecture 1 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 2 Pyruvate Oxidation Glycolysis 2 GLUCOSE DG′° = –17 kcal/mol (–73 kJ/mol) Respiration (+ 6 O2) DG′° = –678 kcal/mol 6 CO2 + 6 H2O (–2,840 kJ/mol) Occurs in three major • Process in which cells consume O2 and produce CO2 stages: • Provides more energy (ATP) per glucose than 1) Pyruvate oxidation (acetyl glycolysis CoA production) • Process is slower than glycolysis 2) acetyl CoA oxidation (CO2 • Evolutionary origin: developed about 2,500 mya production) (TCA cycle) • Used by animals, plants, and many microorganisms • Other fuel sources (lipids and AA) converge on respiration. 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. †Except succinate dehydrogenase, which is located in the inner membrane Inner membrane Outer membrane Cristae Matrix Inter-membrane space 3 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. PDC 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 4 Pyruvate Oxidation PDC is a large (up to 10 MDa) multienzyme complex. -pyruvate dehydrogenase (E1) -dihydrolipoyl transacetylase (E2) -dihydrolipoyl dehydrogenase (E3) 500 Å Cryo-Electron Microscopy E2 complex PDC Nobel Prize for Chemistry in 2017 • 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. Pyruvate Oxidation Box in a box 12; two at each face 24; two at each edge; 3 at each corner E1 The TPP is bound at the interface between the aand b-subunits of the enzyme 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. 5 Pyruvate Oxidation O CH 3 C E1 R–E2 ❺ H E2 R– :B–E2 E2–AH E2 R– ❹ Enzyme 1 • Steps 1-3: Decarboxylation of pyruvate to an enol • Step 4: Acylation of enol/carbanion to a thio-hemiacetal •Step 5: Base deprotonates to form a thioester and release TPP. Hydroxyethyl TPP 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: Base deprotonates to form a thioester and release TPP. Hydroxyethyl TPP 6 Pyruvate Oxidation Coenzyme A Enzyme 2 • Formation of acetyl-CoA: simple thio-ester exchange 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 7 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 Pyruvate Oxidation DE°’ = E°’ (reduction) – E°’ (oxidation) DE°’ = E°’ (NAD+) – E°’ (FAD) = –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 *from Maeda-Yorita et al., (1994) Biochem. 33, 6213 8 Pyruvate Oxidation DE°’ = E°’ (reduction) – E°’ (oxidation) DE°’ = E°’ (NAD+) – E°’ (FAD) = –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 If use S-S, with E°’ (S–S) = –0.34 V, DG°’ = –0.92 kcal/mol *from Maeda-Yorita et al., (1994) Biochem. 33, 6213 Pyruvate Oxidation 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 9 Pyruvate Oxidation E196, E224, E324 E2 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+ Fates of Acetyl CoA (HMG-CoA) Amino acids 1. Oxidation 2. Lipid metabolism 3. Amino acid metabolism 10 Clinical Correlations Physiological consequences of Km values Two enzymes play a key role in the metabolism of alcohol. Some people respond to alcohol consumption with facial flushing and rapid heart beat, symptoms caused by excessive amounts of acetaldehyde in the blood. There are two different acetaldehyde dehydrogenases in most people, one with a low KM value and one with a high KM value. The “low-KM enzyme” is genetically inactivated in some individuals. The “high-KM enzyme” cannot process all of the acetaldehyde, and so some acetaldehyde appears in the blood. Clinical Correlations Warburg Effect: Glycolysis Occurs at Elevated Rates in Tumor Cells • Tumor cells often grow faster than angiogenesis allows growth of capillaries to aerate them, resulting in anaerobic metabolism. • Tumor cells increased expression of LDH, and many glycolytic enzymes, in particular an isozyme of pyruvate kinase (M2), a low activity embryonic for. • Use to generate intermediates for biosynthesis • Compounds that inhibit key steps in glycolysis can kill cancer cells by limiting energy production. 11