Protection of radical intermediates at the active site of adenosylcobalamin-dependent methylmalonyl-CoA mutase

Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the intercon version of methylmalonyl-CoA and succinyl-CoA via radical intermediates gen erated by substrate-induced homolysis of the coenzyme carbon-cobalt bond. F rom the structure of methylmalonyl-CoA mutase it is evident that the deeply buried active site is completely shielded from solvent with only a few pol ar contacts made between the protein and the substrate. Site-directed mutan ts of amino acid His244, a residue close to the inferred site of radical ch emistry, were engineered to investigate its role in catalysis. Two mutants, His244Ala and His244Gln, were characterized using kinetic and spectroscopi c techniques. These results confirmed that Kis244 is not an essential resid ue. However, compared with that of the wild type, k(cat) was lowered by 10( 2)- and 10(3)-fold for the His244Gln and Kis244Ala mutants, respectively, w hile the K-m for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (k(H)/k(T)) for the His244Gln mutant was 1.5 +/- 0.3, and tritium partitioning was now found to be dependent on the substrate used to initiate the reaction, indicating that the rearrange ment of the substrate radical to the product radical was extremely slow. Th e His244Ala mutant underwent inactivation under aerobic conditions at a rat e between 1 and 10% of the initial rate of turnover. The crystal structure of the His244Ala mutant, determined at 2.6 Angstrom resolution, indicated t hat the mutant enzyme is unaltered except for a cavity in the active site w hich is occupied by an ordered water molecule. Molecular oxygen reaching th is cavity may lead directly to inactivation. These results indicate that Hi s244 assists directly in the unusual carbon skeleton rearrangement and that alterations in this residue substantially lower the protection of reactive radical intermediates during catalysis.