III. Metabolism The Citric Acid Cycle

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1 Department of Chemistry and Biochemistry University of Lethbridge Biochemistry 3020 III. Metabolism The Citric Acid Cycle Cellular Respiration Cellular respiration is the step-wise release of energy from glucose, Fatty acids an some amino acids. Energy from these reactions is used to synthesize ATP molecules. This is an aerobic process that requires oxygen (O 2 ) and gives off carbon dioxide (CO 2 ). It involves the complete breakdown of glucose to carbon dioxide and water. 1

2 Catabolism of Proteins, Fats, and Carbohydrates Cellular respiration involves three main phases: Carbon skeletons of organic fuel molecules are degraded to acetyl groups in acetyl-coa Catabolism of Proteins, Fats, and Carbohydrates Cellular respiration involves three main phases: Carbon skeletons of organic fuel molecules are degraded to acetyl groups in acetyl-coa Oxidation of acetyl groups in the citric acid cycle 2

3 Catabolism of Proteins, Fats, and Carbohydrates Cellular respiration involves three main phases: Carbon skeletons of organic fuel molecules are degraded to acetyl groups in acetyl-coa Oxidation of acetyl groups in the citric acid cycle Electrons carried by NADH and FADH 2 are funneled into the respiratory chain. Catabolism of Proteins, Fats, and Carbohydrates The citric acid cycle is called the Krebs cycle or the tricarboxylic and is the hub of the metabolic system. It accounts for the majority of carbohydrate, fatty acid and amino acid oxidation. It also accounts for a majority of the generation of these compounds and others as well. Amphibolic - acts both catabolically and anabolically 3

4 History By 1930 it was established that the addition of lactate, acetate succinate, malate, α-ketoglutaric acid (dicarboxylic acids) and citrate and isocitrate (tricarboxylic acids) when added to muscle mince that they stimulated oxygen consumption and release of CO Albert Szent-Gyorgyi showed that succinate fumarate Malate oxaloacetate Carl Martius and Franz Knoop showed citrate cis-aconitate isocitrate α ketoglutarate succinate fumarate malate oxaloacetate 4

5 History Martius and Knoop showed that pyruvate and oxaloacetate could form citrate non-enzymatically by the addition of peroxide under basic conditions. Krebs showed that succinate is formed from fumarate, malate or oxaloacetate. This is interesting since it was shown that the other way worked as well!! Pyruvate can form citrate enzymatically Pyruvate + oxaloacetate citrate + CO 2 The interconversion rates of the intermediates was fast enough to support respiration rates. Catabolism of Proteins, Fats, and Carbohydrates 5

6 The Citric Acid Cycle Enzymes are Found in the Matrix of the Mitochondria Substrates have to flow across the outer and inner parts of the mitochondria 6

7 Pyruvate is Oxidized to Acetyl-CoA and CO 2 Combined dehydrogenation and decarboxylation of pyruvate requires the sequential action of three different enzymes and five different coenzymes. Coenzyme A Nathan Kaplan and Fritz Lipmann discovered Coenzyme A Ochoa and Lynen showed that acetyl-coa was intermediate from pyruvate to citrate. 7

8 The Pyruvate Dehydrogenase Complex Requires Five Coenzymes Nicotinamide adenine dinucleotide (NAD) Thiamin pyrophosphate (TPP) Flavin adenine dinucleotide (FAD) Coenzym A (CoA) and Lipoate The lipoyllysl moiety acts as a carrier of both hydrogen and an acetyl group. The Pyruvate Dehydrogenase Complex The PDH complex contains three enzymes, present in multiple copies. Number varies among species. E. coli yeast Pyruvate dehydrogenase -- E Dihydrolipoyl transacetylase -- E Dihydrolipoyl dehydrogenase -- E Molecular weight of 4,600,000 Da 50 nm in diameter Lipoate is connected to E 2 8

9 Structure Cryo-EM reconstruction of PDH from bovine kidney Structure E 2 consits of three types of domains linked by short polypeptide linkers. 9

10 Structure and Mechanism Oxidative decarboxylierung of pyruvate to acetyl-coa. Step 1 is rate limiting and responsible for substrate specificity. Structure and Mechanism Why such a complex set of enzymes? Oxidative decarboxylierung of pyruvate to acetyl-coa. Step 1 is rate limiting and responsible for substrate specificity. 10

11 Why such a complex set of enzymes? 1. Enzymatic reactions rates are limited by diffusion, with shorter distance between subunits an enzyme can almost direct the substrate 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. Arsenic Compounds are Poisonous O - OH As OH + HS HS R O - As S S R As(III) compounds, such as arsenit (AsO 3 3- ) and organic arsenicals, are toxic because they covalently bind sulfhydryl compounds. Vicinal (adjacent) sulfhydryld form bidentate adducts 11

12 Structure and Mechanism Mechanism of Dihydrolipoyl Dehydrogenase. More complicated than expected: 1. The Spectrum of oxidized dihydrolipoamide dehydrogenase is unaffected by arsenite. 2. When NADH reacts with the oxidized enzyme in the presence of arsenite, it forms an enzymatically inactive species. 3. 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. The spectrum of the arsenite-inactivated enzyme (2.) indicates that its FAD prosthetic group is fully oxidized. Explanation? 12

13 Mechanism of Dihydrolipoyl Dehydrogenase. Oxidized dehydrolipoamide DH has an additional electron acceptor. The arsenit specificity 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 Catalytic Reaction Cycle of Dihydrolipoyl DH 13

14 The Eight Steps of the Citric Acid Cycle Reactions of the Citric Acid Cycle Overall reaction 3NAD + + FAD + GDP + P i + acetyl-coa 3NADH + FADH + GTP + CoA + 2CO 2 Citric acid cycle is central to the energy-yielding metabolism, but it also produces 4- and 5-corbon precursors for various products. Replenishing (anaplerotic) reactions are needed to keep the cycle going! 14

15 Reactions of the Citric Acid Cycle Step 1 Formation of Citrate by condensation of oxaloacetate and acetyl-coa, catalyzed by citrate synthase. Citroyl-CoA is formed as an intermediate Free energy change has to be large in order to the low concentration of oxaloacetate. Reactions of the Citric Acid Cycle Step 1 Structure of citrate synthase from G. galus mitochondria Open Closed PDBid 5CSC PDBid 5CTS Oxaloacetate binds first and induces large Conformational change creates binding site for Acetyl-CoA 15

16 Structure of Citrate Synthase Reactions of the Citric Acid Cycle Step 1 Mechanism of citrate synthase reaction 1. 16

17 Reactions of the Citric Acid Cycle Step 1 Mechanism of citrate synthase reaction 2. Reactions of the Citric Acid Cycle Step 1 Mechanism of citrate synthase reaction 17

18 Reactions of the Citric Acid Cycle Step 1 Mechanism of citrate synthase reaction Where are the residues in the X-Ray structure? Reactions of the Citric Acid Cycle Step 2 Formation Isocitrate via cis-aconitate Aconitase can promote binding of H 2 O to the enzyme bound cis-aconitate in two ways citarte and isocitrate Equilibrium mixture at ph 7.4 and 25 C contains <10% Isocitrate. 18

19 Reactions of the Citric Acid Cycle Step 2 Mechanism of aconitase Aconitase contains an iron-sulfur center, it acts both in the binding of substrate and the catalytic addition / removal of H 2 O. The formed cis-aconitase usually does not dissociate. PDBid 1B0J Reactions of the Citric Acid Cycle Step 3 Oxidation of Isocitrate to α-ketoglutarate and CO 2 Mn 2+ in the active site interacts with the carbonyl group of intermediate oxalosuccinate and stabilizes the transiently formed. Two different isocitrate DH : a NAD + and a NADP + dependent form. NAD + form in mitochondrial matrix NADP + form in mitochondria and cytosol 19

20 Reactions of the Citric Acid Cycle Step 4 Oxidation of α-ketoglutarate to succinyl-coa and CO 2 The mechanism is identical to the pyruvate dehydrogenase reaction. α-ketoglutarate dehydrogenase complex is very similar to the pyruvate dehydrogenase complex (homologs of E 1, E 2, and E 3 ). It also contains TPP, E 2 bound lipoat, FAD, NAD and CoA. E 3 is identical in both complexes. What about specificity? Reactions of the Citric Acid Cycle Step 5 Conversion of Succinyl-CoA to Succinate The Energy of the thioester bond drives the formation of a phosphoanhydride bond in GTP. 20

21 Reactions of the Citric Acid Cycle Step 5 Mechanism of succinyl-coa synthetase Succinyl-CoA binds to the Enzyme and a phosphoryl group replaces the CoA of succinyl-coa. A high-energy acyl phosphate is formed. Reactions of the Citric Acid Cycle Step 5 Mechanism of succinyl-coa synthetase Succinyl phosphate donates its phosphoryl group to a His residue on the enzyme. The high-energy phosphohistidyl enzyme trabsfers the phosphoryl group to GDP The formed GTP can be converted to ATP by nucleoside diphosphate kinase. 21

22 Reactions of the Citric Acid Cycle Step 5 Mechanism of succinyl-coa synthetase Reactions of the Citric Acid Cycle Step 5 Structure of succinyl-coa synthetase Two subunits: α SU (32 kda) His 246 is phosphorylated β SU (42 kda) confers ATP/GTP specificity Active site is on the SU interface power helices PDBid 1SCU E.coli 22

23 Reactions of the Citric Acid Cycle Step 6 Oxidation of Succinate to Fumarate Succinate dehydrogenase Eukaryotic succinate dehydrogenase is tightly bound to the inner mitochondrial membrane; prokaryotes plasmamembrane. Reactions of the Citric Acid Cycle Step 6 Oxidation of Succinate to Fumarate Succinate dehydrogenase Succinate dehydrogenase contains several iron-sulfur centers that mediate the flow of electrons from succinate via FAD to the respiratory chain and finally to O 2. 23

24 Reactions of the Citric Acid Cycle Step 6 Oxidation of Succinate to Fumarate Succinate dehydrogenase Succinate dehydrogenase contains several iron-sulfur centers that mediate the flow of electrons from succinate via FAD to the respiratory chain and finally to O 2. Malonate, an analog of succinate strongly inhibits succinate dh. normally not present in cells Reactions of the Citric Acid Cycle Step 7 Hydration of Fumarate to Malate Fumarase Highly stereospecific only catalyzes trans double bond hydration and vice versa. no substrates 24

25 Reactions of the Citric Acid Cycle Step 8 Oxidation of Malate to Oxaloacetate L-malate dehydrogenase L-malate is oxidized to oxaloacetate equilibrium lies for on the left. Why can that be? Reactions of the Citric Acid Cycle 25

26 Energy Conserved in the Cycle Yield = 32 x 30.5 kj/mol = 976 kj/mol Oxidation of glucose = 2,840 kj/mol Why is Oxidation of Acetate So Complicated? It is a hub of intermediary metabolism; In aerobic organisms it serves in catabolic and anabolic processes. amphibolic pathway -oxidative catabolism -production of biosynthetic precursers. Intermediates removed form the Cycle are replenished by anaplerotic reactions. incomplete CAC in anaerobic bacteria no α-ketoglutarate dehydrogenase Under stady state conditions (normal) intermediate concentrations remain constant 26

27 Citric Acid Cycle in Anabolism Regulation of the Citric Acid Cycle In mammals, allosteric regulation is complemented by covalent protein modification. E 1 of PDH complex can be covalently modified at a specific Ser residue by a specific protein kinase part of the mammalian PDH inactivation of PDH complex This kinase is allosterically activated by ATP The Citric Acid Cycle Is regulated at Its 3 Exergonic Steps. 27

28 Regulation of the Citric Acid Cycle In mammals, allosteric regulation is complemented by covalent protein modification. Why Calcium? E 1 of PDH complex can be covalently modified at a specific Ser residue by a specific protein kinase part of the mammalian PDH inactivation of PDH complex This kinase is allosterically activated by ATP The Citric Acid Cycle Is regulated at Its 3 Exergonic Steps. And Another One - The Glyoxylate Cycle In many organisms other than vertebrates, the glyoxylate cycle serves as mechanism for converting acetate to carbohydrate. The glyoxylate cycle produces four-carbon compounds from acetate. In plant the glyoxylate cycle enzymes are found in membrane-bounded organelles Glyoxysomes Found in lipid rich seeds during germination, before glucose from photosynthesis is available. 28

29 The Glyoxylate Cycle Citrate synthase, aconitase, and malate dehydrogenase are isozymes of the CAC enzymes. Isocitrate lyase and malate synthae are unique to the cycle Relationship Between the Glyoxylate And the Citric Acid Cycle 29

30 Coordinated Regulation Sharing common intermedeates requires coordinated regulation. Isocitrate is at the branching point between the glyoxylate and the CA cycle Isocitrate DH is regulated by covalent modification (specific protein kinase) E. coli has the full complement of enzymes and therefore grows on acetate as the sole carbon source. Reading Chapter 17 Fatty Acid Catabolism 30