Rotation of the exo-Methylene Group of 2-Methyleneglutarate Catalyzed by Coenzyme B_(12)-Dependent 2-Methyleaeglutarate Mutase from Clostridium barken

摘要

2-Methyleneglutarate mutase from Clostridium barken is a cpenzyme B_(12)-dependent enzyme that catalyses a remarkable carbon skeleton rearrangement, namely the equilibration of 2-methyleneglutarate la with (R)-3-methylitaconate [(R)-2-memyl-3-methylenesuccinate 2] (see Scheme 1). This process is believed to be initiated by binding of the substrate to the holoenzyme, which brings about homolysis of the Co--C σ-bond of the coenzyrae B_(12) (adenosylcobalamin) yielding cob(II)alamin and a methylene (5'-deoxyadenosyl) radical. The latter abstracts H_(Re) from C-4 of 2-methyleneglutarate~(1c) generating the substrate-derived radical 3 shown in Scheme 1. An attractive mechanism for the rearrangement of this species to a product-related radical 4 postulates the intermediacy of a cyclopro-pylcarbinyl radical 5 (see Scheme 1). The rapid interconversion of but-3-enyl and cyclopropylcarbinyl radicals is well known in nonenzymic chemistry, and model systems have been described in support of the mechanism of Scheme 1. We report a critical test of this mechanism. If the energy barrier to rotation about the C-l/methylene bond in 5 is, sufficiently low then a stereospecifically deuteriated specimen of 2-methyleneglutarate (say 1b) would equilibrate with 1c whenincubated with 2-methyleneglutarate mutase holoenzyme. We have found that this is indeed the case.