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The Citric Acid Cycle 11/25/2008 The Citric acid cycle It 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 3NAD+ + FAD + GDP + Pi + acetyl-CoA 3NADH + FADH + GTP + CoA + 2CO2 Overview 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 Pyruvate dehydrogenase A multienzyme complexes are groups of non covalently associated enzymes that catalyze two or more sequential steps in a metabolic pathway. Molecular weight of 4,600,000 Da E. coli Pyruvate dehydrogenase -- yeast E1 24 60 dihydrolipoyl transacetylase --E2 24 60 dihydrolipoyl dehydrogenase--E3 12 12 Why such a complex set of enzymes? 1 Enzymatic reactions rates are limited by diffusion, with shorter distance between subunits a enzyme can almost direct the substrate from one subunit (catalytic site) to another. 2. Channeling metabolic intermediates between successive enzymes minimizes side reactions 3. The reactions of a multienzyme complex can be coordinately controlled Covalent modification of eukaryotic pyruvate dehydrogenase The five reactions of the pyruvate dehydrogenase multi enzyme complex Citrate Synthase O HO CH2 C O O + H3C HO C O O HO CH2 HO C CH2 HO O O OH SCoA Induced fit needs binding of oxaloacetate before Acetyl CoA can bind. O CoAS C OH CoAS CH3 CH2 Acetyl-CoA Proposed intermediate OH O CoAS CH2 CoAS C CH3 Acetonly CoA (ground-state analog) C CH2 O Carboxymethyl-CoA (transition state analog) Aconitase O HO CH2 HO CH2 O O Citrate O HO CH2 O CH C CH2 HO O HO O HC OH OH CH HO HO O Cis-Aconitate HO C H OH O Isocitrate The double bond is placed on the Pro-R arm NAD+- Dependent Isocitrate dehydrogenase NAD+ NADH a-Ketoglutarate dehydrogenase O HO HO NAD+ CO2 CH2 CH2 H2C C HO O O O CH2 NADH CoAS O This enzyme is just like pyruvate dehydrogenase, a multi enzyme complex that is specific for longer CoA derivatives Refresh:The five reactions of the pyruvate dehydrogenase multi enzyme complex Succinyl-CoA Synthetase or succinate thiokinase ← Note symmetry Succinate dehydrogenase HO O CH CH2 O + 2e- + 2H+ CH CH2 HO O HO HO O The FAD on the enzyme itself is reduced Succinate dehydrogenase is the only membrane bound enzyme in the citrate cycle O H3CO CH3 Succ dh--FADH2 + CH2 H3CO n O Ubiquinone or Coenzyme Q n = 6-10 CH3 Oxidized form OH H3CO CH3 CH2 n H3CO OH CH3 Reduced form Fumarase Malate dehydrogenase O HO NADH H2 C H C O HO H2 C OH C O NAD+ HO O HO O Regulation of the citric acid cycle Standard free energy changes in the citric acid cycle Reaction 1 2 3 4 5 6 7 8 Enzyme Citrate synthase Aconitase Isocitrate dh a-KG dh Succinyl-CoA synthase Succinate dh Fumarase Malate dh DG' -31.5 ~5 -21 -33 -20.1 +6 -3.4 +29.7 DG' Negative ~0 Negative Negative ~0 ~0 ~0 ~0 The points of regulation of the cycle Citric acid cycle intermediates are always in flux A single molecule of glucose can potentially yield ~38 molecules of ATP No Lecture Thursday 11/27/08 Thanksgiving Next Lecture Tuesday 12/02/08 Pentose phosphate pathway