Thermodynamic and kinetic characterization of Co-C bond homolysis catalyzed by coenzyme B(12)-dependent methylmalonyl-CoA mutase.

Methylmalonyl-CoA mutase is a member of the family of coenzyme B(12)-dependent isomerases and catalyzes the 1,2-rearrangement of methylmalonyl-CoA to succinyl-CoA. A common first step in the reactions catalyzed by coenzyme B(12)-dependent enzymes is cleavage of the cobalt-carbon bond of the cofactor, leading to radical-based rearrangement reactions. Comparison of the homolysis rate for the free and enzyme-bound cofactors reveals an enormous rate enhancement which is on the order of a trillion-fold. To address how this large rate acceleration is achieved, we have examined the kinetic and thermodynamic parameters associated with the homolysis reaction catalyzed by methylmalonyl-CoA mutase. Both the rate and the amount of cob(II)alamin formation have been analyzed as a function of temperature with the protiated substrate. These studies yield the following activation parameters for the homolytic reaction at 37 degrees C: DeltaH(f)() = 18.8 +/- 0.8 kcal/mol, DeltaS(f)() = 18.2 +/- 0.8 cal/(mol.K), and DeltaG(f)() = 13.1 +/- 0.6 kcal/mol. Our results reveal that the enzyme lowers the transition state barrier by 17 kcal/mol, corresponding to a rate acceleration of 0.9 x 10(12)-fold. Both entropic and enthalpic factors contribute to the observed rate acceleration, with the latter predominating. The substrate binding step is exothermic, with a DeltaG of -5.2 kcal/mol at 37 degrees C, and is favored by both entropic and enthalpic factors. We have employed the available kinetic and spectroscopic data to construct a qualitative free energy profile for the methylmalonyl-CoA mutase-catalyzed reaction.