Tritium isotope effects in adenosylcobalamin-dependent methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase from Propionibacterium shermanii is an adenosylcobalamin-dependent enzyme which catalyzes the reversible isomerization of methylmalonyl-Coa and succinyl-CoA. The rate of tritium loss from 5'-[H-3]adenosylcobalamin during the enzymic reaction and the relative rates of tritium appearance in substrate and product were examined. Upon the addition of methylmalonyl-CoA to a solution of holoenzyme, tritium was completely released from the cofactor within about 500 ms. No tritium was found either bound to the enzyme or released into the water. The radioactivity was found in methylmalonyl-CoA and succinyl-Coa in a constant ratio of 1 to 3, which did not change during the first 300 ms of the reaction. Upon the addition of succinyl-CoA to a solution of holoenzyme, tritium was released at essentially the same rate, and the radioactivity was found in methylmalonyl-CoA and succinyl-Coa in the identical constant ratio of 1 to 3. The tritium isotope effect on the enzyme-catalyzed hydrogen transfer, measured using C-14-labeled methylmalonyl-CoA as substrate, was k(H)/k(T) = 4.9. This low value shows that hydrogen transfer is only partly rate limiting and that at least one subsequent slow step, such as product release, contributes substantially to the overall reaction velocity. The identical partitioning of tritium, regardless of the substrate used, shows that the rearrangement of the substrate radical into the product radical is not rate limiting. The very low tritium isotope effect and the fact that all the tritium is found bound either to the CoA esters or to the cofactor make it very unlikely that a protein radical is an intermediate in the methylmalonyl-CoA mutase-catalyzed rearrangement.