Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila

The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-¹⁴C] acetyl-coenzyme A (CoA) (carbonyl group) with ¹²CO as well as the exchange of [3'-³²P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: K(m) (acetyl-CoA)= 200 μM, V(max) = 15 min^-1. CoA is a potent inhibitor of this exchange (K(i) = 25 μM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria.