Evidence for a 1,2 shift of a hydrogen atom in a radical intermediate of the methylmalonyl-CoA mutase reaction

An excellent substrate of methylmalonyl-CoA mutase, methylmalonyl-carba-(de thia) coenzyme A (methylmalonyl-CH2-CoA), was synthesized by a chemoenzymat ic method and its alpha-proton was exchanged with deuterium by long-term in cubation in deuterium oxide at pH 6.9. After addition of highly purified ep imerase-free methylmalonyl-CoA mutase the enzymatic rearrangement was monit ored by H-1 NMR spectroscopy. Already in the initial phases of the reaction only 72% of the produced succinyl-CH2-CoA was monodeuterated, while unlabe led and geminally dideuterated species, 14% of each, were also formed. Afte r the addition of more enzyme the equilibrium (methylmalonyl-CoA:succinyl-C oA = 1:20) was quickly established, while the proportion of unlabeled succi nyl-CH2-CoA rose to 30% and the geminally dideuterated species were slowly transformed to vicinally dideuterated ones. After 19 h of incubation the ra tio of the unlabeled, monodeuterated. and dideuterated species was roughly 1:1:1 while no appreciable deuterium incorporation from the solvent occurre d. The unexpected disproportionation of deuterium can be best explained by a 1,2 shift of a hydrogen atom in the succinyl-CH2-CoA radical intermediate competing with the hydrogen transfer from 5'-deoxyadenosine. A precedence for such a hydrogen shift in a radical was previously observed only in the mass spectrometer and was supported by ab initio calculations. (C) 2000 Aca demic Press.