Proton transfer from histidine 244 may facilitate the 1,2 rearrangement reaction in coenzyme B-12-dependent methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase is an adenosylcobalamin-dependent enzyme that cata lyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA This react ion results in the interchange of a carbonyl-CoA group and a hydrogen atom on vicinal carbons. The crystal structure of the enzyme reveals the presenc e of an aromatic cluster of residues in the active site that includes His-2 44, Tyr-243, and Tyr-89 in the large subunit, Of these, His-244 is within h ydrogen bonding distance to the carbonyl oxygen of the carbonyl-Cob moiety of the substrate. The location of these aromatic residues suggests a possib le role for them in catalysis either in radical stabilization and/or by dir ect participation in one or more steps in the reaction. The mechanism by wh ich the initially formed substrate radical isomerizes to the product radica l during the rearrangement of methylmalonyl-CoA to succinyl-CoA is unknown. Ab initio molecular orbital theory calculations predict that partial proto n transfer can contribute significantly to the lowering of the barrier for the rearrangement reaction. In this study, we report the kinetic characteri zation of the H244G mutant, which results in an acute sensitivity of the en zyme to oxygen, indicating the important role of this residue in radical st abilization Mutation of His-244 leads to an similar to 300-fold lowering in the catalytic efficiency of the enzyme and loss of one of the two titratab le pK(a) values that govern the activity of the wild type enzyme. These dat a suggest that protonation of His-244 increases the reaction sate in wild t ype enzyme and provides experimental support for ab initio molecular orbita l theory calculations that predict rate enhancement of the rearrange ment r eaction by the interaction of the migrating group with a general acid. Howe ver, the magnitude of the rate enhancement is significantly lower than that predicted by the theoretical studies.