Electron paramagnetic resonance studies of the methylmalonyl-CoA mutase reaction. Evidence for radical intermediates using natural and artificial substrates as well as the competitive inhibitor 3-carboxypropyl-CoA.

The substrate-dependent homolysis of the cobalt-carbon bond and generation of organic radicals in the coenzyme-B12-methylmalonyl-CoA-mutase complex have been demonstrated by EPR measurements. Both the natural substrate methylmalonyl-CoA, its 13C-substituted analogue and the non-hydrolysable synthetic substrates succinyl-dethia(carba)-CoA, succinyl-dethia(dicarba)-CoA and 4-carboxy-2-oxo-butyl-CoA induced similar but not identical EPR signals. 3-Carboxypropyl-CoA, a novel competitive inhibitor, has been synthesised. Its Ki value of 89 +/- 6 microM was in the same range as the Km of succinyl-CoA. Using [5'-3H]adenosylcobalamin, an enzyme-dependent tritium transfer to the inhibitor has been shown. The enzyme-coenzyme-inhibitor complex also exhibited EPR signals that were less structured and less intensive than the corresponding signals with active substrates. These results prove that the inhibitor also induces cobalt-carbon bond homolysis and undergoes reversible hydrogen transfer but not rearrangement.