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-B-12-methylmalonyl-CoA-mutase complex have been demonstrated by EPR measurements. Both the natural substrate methylmalonyl-CoA, its C-13-substituted analogue and the nonhydrolysable 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 K-i value of 89+/-6 mu M was in the same range as the K-m of succinyl-CoA. Using [5'-H-3] 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.