Insights into the kinetics and reaction mechanism of B(12)-dependent methylmalonyl-CoA mutase

B12-dependent methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA in the degradation of odd-chain fatty acids, branched-chain amino acids and cholesterol to energetically useful metabolites. Dysfunction of methylmalonyl-CoA mutase is inherited as an autosomal recessive disorder leading to methylmalonic aciduria, manifested by elevated levels of methylmalonic acid. The first step in AdoCbl-dependent enzyme reactions is believed to be the homolytic fission of the Co-C bond of the cofactor to generate a carbon-centered deoxyadenosyl radical and an unpaired electron on the cobalt atom. Methylmalonyl-CoA mutase accelerates the homolysis step 1012-fold in the presence of substrate. The main objective of this study was to evaluate the contribution of select active site residues, that interact with the cofactor and the substrate, to the tremendous enhancement of the homolysis rate and to the stabilization of the highly reactive radical intermediates formed during catalysis. To this end we have examined the effect of the dimethylbenzimidazole displacement from the cobalt by a histidine residue, H610 upon binding of the cofactor to the mutase. Our mutational studies show that H610 plays a structural role in organizing the cofactor binding site and in catalyzing the dimethylbenzimidazole displacement, in a step preceding cofactor binding. We have also evaluated the contribution of Y89 residue to the enhancement of the Co-C bond homolysis rate. From kinetic studies on the mutants Y89F and Y89A we have estimated that 103-104-fold contribution to the homolysis rate is brought about by this residue. We have also examined the contribution of R207, which is in hydrogen-bond contact with the substrate carboxylate to the mutase catalyzed reaction, by evaluating the properties of the R207Q mutant. Loss of this hydrogen-bonding interaction has a profound effect in catalysis and substrate binding. We also reported in this study the first example of suicide inactivation of methylmalonyl-CoA mutase by an undesired internal electron transfer from the cob(II)alamin intermediate, in an oxygen-independent process. Together these studies have enhanced our understanding of how specific active site residues modulate binding or catalysis and suppress unwanted side reactions in methylmalonyl-CoA mutase