Chapter 9 Chem 341 Suroviec Fall 2016

I. Citric Cycle Overview 8 reactions Oxidizes acetyl group of Acetyl CoA to 2 CO2

I. General Features Circular Pathway oxidizes acetyl groups from many sources Net reaction In eukaryotes all enzymes of CAC are located in mitochondria 1 CO2 produced in 1 round of the cycle Oxidation of acetyl groups to 2 CO2 requires transfer of 4 pair of electrons

II. Synthesis of Acetyl CoA Pyruvate Dehydrogenase: Multienzyme Complex Group of non covalently associated enzymes that catalyze 2+ sequential steps in metabolic pathway Formed from pyruvate through oxidative decarboxylation Pyruvate dehydrogenase (E1) Dihydrolipoyl transacetylase (E2) Dihydrolipoyl dehydrogenase (E3) 24 E2 proteins associated as trimers at the corners of cube 24 E1 proteins form dimers that associate with E2 core along the 12 edges. The 12 E3 proteins form dimers that attach to the 6 faces of E2 cube c) Combining a) and b) forms a 60 subunit complex

Section 9.2: Citric Acid Cycle Decarboxylation Action of lipoic acid Figure 9.10 Reactions Catalyzed by the Pyruvate Dehydrogenase Complex From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

B. Pyruvate Dehydrogenase Overall Reaction Pyruvate dehydrogenase (E1) A TPP requiring enzyme TPP acts as electron sink in the reaction

2. Lipoamide Hydroxyethyl group transferred to E2 Lipoamide uses lysine Cyclic disulfide reversibly reduced

3. E2 transesterification Yields acetyl CoA and dihydrolipoamide-E2 4. Regenerate E2 E3 becomes reduced Regenerates E2 Disulfide interchange reaction

5. Reoxidize E3 Reoxidize E3

Section 9.2: Citric Acid Cycle Figure 9.15 Citrate Metabolism From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 9.2: Citric Acid Cycle Figure 9.15 Citrate Metabolism From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

III. Enzymes of CAC Citrate synthase Aconitase Isocitrate dehydrogenase a-ketoglutarate dehydrogenase: Succinyl CoA synthetase Succinate dehydrogenase Fumerase Malate dehydrogenase

III. Enzymes of the CAC Citrate Synthase Catalyzes the condensation of acetyl-CoA and oxaloacetate Free enzyme is a dimer Active site closes when oxaloacetate binds Conformational changes seals oxaloacetate in binding site and shuts out the solvent

C. NAD+ Dependant Isocitrate Dehydrogenase B. Aconitase Catalyzes reversible isomerization of citrate to isocitrate C. NAD+ Dependant Isocitrate Dehydrogenase Catalyzes oxidative decarboxylation of isocitrate to a-ketogluterate

D. a-ketoglutarate dehydrogenase Catalyzes oxidative decarboxylation of a-ketogluterate Is a multienzyme complex E1: a-ketoglutarate dehydrogenase E2: dihydrolipoyl transsuccinylase E3: dihydrolipoyl dehydrogenase

E. Succinyl-CoA Synthetase Cleaves “high-energy” succinyl-CoA to synthesis of GTP Reaction almost energy neutral.

F. Succinate Dehydrogenase Catalyzes stereospecific dehydrogenation of succinate to fumerate Inhibited by malonate

H. Malate dehydrogenase G. Fumerase Catalyzes the hydration of double bond of fumarate to form malate H. Malate dehydrogenase Regeneration of oxaloacetate

Availibity of substrates Need for CAC intermediates Demand for ATP IV. Regulation of CAC Availibity of substrates Need for CAC intermediates Demand for ATP

A. Regulation of pyruvate decarboxylation Product inhibition by NADH and acetyl-CoA NADH, acetyl-CoA compete with NAD+ and CoA for binding sites Drive with E2 and E3 Covalent modification by phosphorylation/dephosphorylation of E1

B. Rate-Controlling Enzymes Flux of metabolites through the CAC is proportional to the rate of cellular oxygen consumption 3 main mechanisms Substrate availability Product inhibition Competitive feedback inhibition

B. Rate-Controlling Enzymes Regulators are acetyl-CoA, oxaloacetate, NADH Flux varies with substrate concentration

V. Pathways that use CAC intermediates Glucose biosynthesis Fatty acid synthesis Amino Acid synthesis