title: When a spectator turns killer: suicidal electron transfer from cobalamin in methylmalonyl-CoA mutase.
abstract: Methylmalonyl-CoA mutase belongs to the class of adenosylcobalamin (AdoCbl)-dependent carbon skeleton isomerases and catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. In this study, we have evaluated the contribution of the active site residue, R207, in the methylmalonyl-CoA mutase-catalyzed reaction. The R207Q mutation results in a 10(4)-fold decrease in k(cat) and >30-fold increase in the K(M) for the substrate, methylmalonyl-CoA. R207 and the active site residue, Y89, are within hydrogen bonding distance to the carboxylate of the substrate. In the closely related isomerase, isobutyryl-CoA mutase the homologous residues are F80 and Q198, respectively. We therefore characterized the ability of the double mutant (Y89F/R207Q) of methylmalonyl-CoA mutase as well as of the single mutants (Y89F and R207Q) to catalyze the rearrangement of n-butyryl-CoA to isobutyryl-CoA. While none of the mutant enzymes is capable of isomerizing these substrates, the R207Q (single and double) mutants exhibited irreversible inactivation upon incubation with either n-butyryl-CoA or isobutyryl-CoA. The two products observed during inactivation under both aerobic and strictly anaerobic conditions were 5'-deoxyadenosine and hydroxocobalamin, which suggested internal electron transfer from cob(II)alamin to the substrate or the 5'-deoxyadenosyl radical. Deuterium transfer from substrate to deoxyadenosine demonstrated that the substrate radical is formed and is presumably the acceptor in the electron-transfer reaction from cob(II)alamin. These studies provide evidence for the critical role of active site residues in controlling radical reactivity and thereby suppressing inactivating side reactions.
authors.affiliation: Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
authors.forename: Monica D
authors.initials: MD
authors.lastname: Vlasie
authors.forename: Ruma
authors.initials: R
authors.lastname: Banerjee
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002264:Carboxylic Acids / Q000737:chemistry; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003038:Cobamides / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D004579:Electron Transport; D004583:Electrons; D006860:Hydrogen Bonding; D007700:Kinetics; D008765:Methylmalonyl-CoA Mutase / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D014466:Ultraviolet Rays; D014805:Vitamin B 12 / Q000737:chemistry*
chemical_list: D000214:Acyl Coenzyme A; D002264:Carboxylic Acids; D003038:Cobamides; C050106:isobutyryl-coenzyme A; C024343:butyryl-coenzyme A; D003903:Deuterium; D008765:Methylmalonyl-CoA Mutase; C000913:cobamamide; D014805:Vitamin B 12