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Department of Chemistry and Biochemistry University of Lethbridge Biochemistry 3300 III. Metabolism The Citric Acid Cycle Biochemistry 3300 Slide 1 Cellular Respiration (combustion) Cellular respiration is the step-wise release of energy from glucose, fatty acids and (some) amino acids. •Efficient aerobic process that requires oxygen and produces carbon dioxide. Energy from these reactions is used to synthesize ATP molecules. Involves the complete oxidation of glucose to carbon dioxide and water. C atoms* (glucose) C atom (CO2) Biochemistry 3300 Oxidation Number 4 0 Slide 2 Catabolism of Proteins, Fats, and Carbohydrates Cellular respiration involves three main phases: Phase 1 Carbon skeletons of organic fuel molecules are degraded to acetyl groups that are attached to acetyl-CoA Biochemistry 3300 Slide 3 Catabolism of Proteins, Fats, and Carbohydrates Cellular respiration involves three main phases: Phase 1 Carbon skeletons of organic fuel molecules are degraded to acetyl groups that are attached to acetyl-CoA Phase 2 Oxidation of acetyl groups in the citric acid cycle Biochemistry 3300 Slide 4 Catabolism of Proteins, Fats, and Carbohydrates Cellular respiration involves three main phases: Phase 1 Carbon skeletons of organic fuel molecules are degraded to acetyl groups that are attached to acetyl-CoA Phase 2 Oxidation of acetyl groups in the citric acid cycle Phase 3 Electrons carried by NADH and FADH2 are funneled into the respiratory chain. Biochemistry 3300 Slide 5 Catabolism of Proteins, Fats, and Carbohydrates The citric acid cycle is also called the Krebs cycle or the tricarboxylic acid (TCA) cycle. The citric acid cycle is the “hub” of the metabolic system. - Majority of carbohydrate, fatty acid and amino acid oxidation. - Majority of the generation of these compounds and others. Citric Acid Cycle is amphibolic as it acts both catabolically and anabolically Biochemistry 3300 Slide 6 History By 1930, it was established that some compounds: Carboxylic acids (acetate, lactate) Dicarboxylic acids (succinate, malate, -ketoglutarate) and Tricarboxylic acids (citrate, isocitrate) would stimulate O2 consumption and CO2 production when added to “minced” muscle 1935: Albert Szent-Gyorgyi Succinate → Fumarate → Malate → Oxaloacetate Carl Martius & Franz Knoop Citrate [→ Cis-aconitate] → Isocitrate → -ketoglutarate → Succinate → Fumarate → Malate → Oxaloacetate Biochemistry 3300 Slide 7 History Subsequently, Martius & Knoop showed: Pyruvate and Oxaloacetate can form citrate non-enzymatically (requires peroxide and basic conditions). Odd: oxaloacetate is the product in their pathway! And then Hans Krebs the showed: Succinate is formed from fumarate, malate or oxaloacetate. Odd: These appear to be the reverse reactions! Citric Acid metabolic pathway is a CYCLE !! Biochemistry 3300 Slide 8 Catabolism of Proteins, Fats, and Carbohydrates Glycolysis – cytosol TCA cycle – mitochondria (eucaryotes) Biochemistry 3300 Slide 9 Citric Acid Cycle enzymes are in the mitochondrial matrix Substrates must cross both the outer and inner mitochondrial membrane Biochemistry 3300 Slide 10 Mitochondrial Membrane Particles visualized by EM are “large” protein complexes Inner membrane is rich in large protein complexes Biochemistry 3300 Slide 11 Coenzyme A Nathan Kaplan and Fritz Lipmann discovered Coenzyme A (CoA) Ochoa and Lynen showed that acetyl-CoA is an intermediate in the conversion of pyruvate to citrate. Biochemistry 3300 Slide 12 Pyruvate is oxidized to acetyl-CoA and CO2 Combined dehydrogenation and decarboxylation of pyruvate requires the sequential action of three different enzymes (E1, E2, E3) and five different coenzymes. Biochemistry 3300 Slide 13 Pyruvate Dehydrogenase Complex (PDH) PDH complex contains three subunits, present in multiple copies. Number varies among species. E. coli yeast Pyruvate dehydrogenase -- E1 24 60 Dihydrolipoyl transacetylase -- E2 24 60 Dihydrolipoyl dehydrogenase -- E3 12 12 Molecular weight of 4,600,000 Da ; 50 nm in diameter Lipoate is connected to E2 Biochemistry 3300 Slide 14 Pyruvate Dehydrogenase Complex Requires Five Coenzymes 1) Nicotinamide adenine dinucleotide (NAD+) 2) Thiamin pyrophosphate (TPP) 3) Flavin adenine dinucleotide (FAD) 4) Coenzyme A (CoA) 5) Lipoate The lipoyllysl moiety acts as a carrier of both hydrogen and an acetyl group. Biochemistry 3300 Slide 15 Structure Cryo-EM reconstruction of PDH from bovine kidney Biochemistry 3300 Slide 16 Structure E2 consists of three types of domains linked by short polypeptide linkers. Biochemistry 3300 Slide 17 Structure and Mechanism Oxidative decarboxylation of pyruvate to acetyl-CoA. Step 1 is rate limiting and responsible for substrate specificity. Biochemistry 3300 Slide 18 Structure and Mechanism Decarboxylation of pyruvate and formation of acetyl lipoyllysine Biochemistry 3300 Slide 19 Structure and Mechanism Formation of Acetyl-CoA Biochemistry 3300 Slide 20 Why such a complex set of enzymes? 1. Enzymatic reaction rates are limited by diffusion, with shorter distance between subunits in an enzyme, the substrate can be directed from one subunit (catalytic site) to another. 2. Channeling metabolic intermediates between successive enzymes minimizes side reactions. (Substrate channeling). 3. Local substrate concentration is kept high. 4. The reactions of a multienzyme complex can be coordinately controlled / regulated. Biochemistry 3300 Slide 21 Arsenic Compounds are Poisonous - O S HS OH O- As + As OH S HS R R As(III) compounds, such as arsenite (AsO33-) and organic arsenicals, are toxic because they covalently attach to sulfhydryl compounds. Vicinal (adjacent) sulfhydryls form bidentate adducts (top right) Biochemistry 3300 Slide 22 Structure and Mechanism Arsenite inhibits E3 Biochemistry 3300 Slide 23 Mechanism of Dihydrolipoyl Dehydrogenase. More complicated than expected: 1. Spectra of oxidized dihydrolipoamide dehydrogenase (E3) is unaffected by arsenite. 2. NADH reaction with the oxidized enzyme in the presence of arsenite → forms an enzymatically inactive species. 3. Spectrum of the arsenite-inactivated enzyme (2.) indicates that its FAD prosthetic group is fully oxidized. Recall: The oxidation state of the flavin in a flavoprotein is readily established from its characteristic UV-Vis Spectrum: FAD is intense yellow, whereas FADH2 is pale yellow. Explanation ? Biochemistry 3300 Slide 24 Mechanism of Dihydrolipoyl Dehydrogenase. Oxidized dehydrolipoamide dehydrogenase has an additional electron acceptor. Arsenite inhibition suggests a disulfide as acceptor. See X-Ray structure of dehydrolipoamide DH from P. putida, PDBID 1LVL Catalytic active residues: Cys 43 & 48 , Tyr 181 Biochemistry 3300 Slide 25 Mechanism of Dihydrolipoyl Dehydrogenase. Arsenite target Biochemistry 3300 Slide 26 Catalytic Cycle of Dihydrolipoyl dehydrogenase Biochemistry 3300 Slide 27 Eight Steps of the Citric Acid Cycle NEXT Biochemistry 3300 Slide 28