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.