B12-dependent
methylmalonyl-CoA mutase catalyses the interchange of a
hydrogen atom and the carbonyl-
CoA group on adjacent carbons of
methylmalonyl-CoA to give the rearranged product,
succinyl-CoA. The first step in this reaction involves the transient generation of cofactor radicals by homolytic
rupture of the
cobalt-
carbon bond to generate the deoxyadenosyl radical and
cob(II)alamin. This step exhibits a curious sensitivity to isotopic substitution in the substrate,
methylmalonyl-CoA, which has been interpreted as evidence for kinetic coupling. The magnitude of the isotopic discrimination is large and a
deuterium isotope effect ranging from 35.6 at 20 degrees C to 49.9 at 5 degrees C has been recorded. Arrhenius analysis of the temperature dependence of this
isotope effect provides evidence for quantum tunnelling in this
hydrogen transfer step. The mechanistic complexity of the observed rate constant for
cobalt-
carbon bond homolysis together with the spectroscopically silent nature of many of the component steps limits the insights that can be derived by experimental approaches alone. Computational studies using a newly developed geometry optimization scheme that allows determination of the transition state in the full quantum mechanical/molecular mechanical coordinate space have yielded novel insights into the strategy deployed for labilizing the
cobalt-
carbon bond and poising the resulting deoxyadenosyl radical for subsequent
hydrogen atom abstraction.