Kinetic characterization of the [3'-32P]coenzyme A/acetyl coenzyme A exchange catalyzed by a three-subunit form of the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum.
The ability of acetyl coenzyme A synthesizing carbon monoxide dehydrogenase isolated from Clostridium thermoaceticum to catalyze the exchange of [3'-32P]coenzyme A with acetyl coenzyme A is studied. This exchange is found to have a rate exceeding that of the acetyl coenzyme A carbonyl exchange also catalyzed by CO dehydrogenase ([1-14C]acetyl coenzyme A + CO in equilibrium acetyl coenzyme A + 14CO). These two exchanges are diagnostic of the ability of CO dehydrogenase to synthesize acetyl coenzyme A from a methyl group, coenzyme A, and carbon monoxide. The kinetic parameters for the coenzyme A exchange have been determined: Km(acetyl coenzyme A) = 1500 microM, Km(coenzyme A) = 50 microM, and Vmax = 2.5 mumol min-1 mg-1. Propionyl coenzyme A is shown to be a substrate (Km approximately 5 mM) for the coenzyme A exchange, with a rate 1/15 that of acetyl coenzyme A, but is not a substrate for the carbonyl exchange. CO dehydrogenase capable of catalyzing both these two exchanges, and the oxidation of CO to CO2, is isolated as a complex of molecular weight 410,000 consisting of three proteins in an alpha 2 beta 2 gamma 2 stoichiometry. The proposed gamma subunit, not previously reported as part of CO dehydrogenase, copurifies with the enzyme and has the same molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the disulfide reductase previously separated from CO dehydrogenase in a final chromatographic step.
Ramer
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Raybuck
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Orme-Johnson
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Walsh
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D000106:Acetate-CoA Ligase / Q000032:analysis*; D000105:Acetyl Coenzyme A / Q000032:analysis*; D000214:Acyl Coenzyme A / Q000032:analysis; D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D002845:Chromatography / Q000379:methods; D003013:Clostridium / Q000201:enzymology*; D003065:Coenzyme A / Q000032:analysis*; D003066:Coenzyme A Ligases / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009097:Multienzyme Complexes; D010084:Oxidation-Reduction
D000214:Acyl Coenzyme A; D009097:Multienzyme Complexes; C009061:propionyl-coenzyme A; D000105:Acetyl Coenzyme A; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D003066:Coenzyme A Ligases; D000106:Acetate-CoA Ligase; D003065:Coenzyme A