Evidence that cobalt-carbon bond homolysis is coupled to hydrogen atom abstraction from substrate in methylmalonyl-CoA mutase.

Methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA. It is dependent on the cofactor, coenzyme B12 or adenosylcobalamin, for activity. The first step in this, and other coenzyme B12-dependent reactions, is postulated to be homolysis of the Co-C bond of the cofactor. Methylmalonyl-CoA mutase accelerates the rate of Co-C bond homolysis by a factor of approximately 10(12). The strategy employed by the enzyme for the remarkable labilization of this bond is not known. Using UV-visible stopped-flow spectrophotometry, we demonstrate that the Co-C homolysis rate in the presence of protiated substrate has a rate constant of >600 s(-1) at 25 degrees C. In the presence of [CD3]methylmalonyl-CoA, this rate decreases to 28 +/- 2 s(-1). These results suggest that Co-C bond homolysis is coupled to hydrogen atom abstraction from the substrate and that the intrinsic binding energy of substrate may be a significant contributor to catalysis by methylmalonyl-CoA mutase.