Inactivation of Medium-Chain Acyl-CoA Dehydrogenase by a Metabolite of Hypoglycin: Characterization of the Major Turnover Product and Evidence Suggesting an Alternative Flavin Modification Pathway

Article abstract of DOI:10.1021/ja00058a001

Medium-chain acyl-CoA dehydrogenase (MCAD) is a FAD-dependent enzyme that catalyzes the first step of the fatty acid oxidation cycle.When MCAD is exposed to (methylenecyclopropyl)acetyl-CoA (MCPA-CoA), a metabolite of hypoglycin A and the causative agent of Jamaican vomiting sickness, time-dependent inactivation follows with concomitant bleaching of the active-site FAD.Earlier studies have led to the postulation that the inactivation may involve a spontaneous ring fragmentation induced by a transient α-cyclopropyl radical, and thus suggest a one-electron oxidation pathway.In an effort to find more evidence for the proposed mechanism, we have isolated and characterized the major turnover product, a CoA ester consisting of a disubstituted terminal olefin, an epoxide, and a hydroxymethyl group, from the aerobic incubation mixture of MCPA-CoA and MCAD.Formation of this product may be initiated by trapping the acyclic radical intermediate with O2 to form a transient peroxy radical which, upon receiving one electron from flavin semiquinone followed by an intramolecular epoxidation, gives rise to the observed turnover product.The identification of such a highly oxygenated species as the major turnover product strongly sustains the intermediacy of a ring-opened radical, and as such, the departure from the expected inactivation may directly result from trapping of this radical intermediate by O2.This contention was subsequently substantiated by observing that the partition ratio is nearly 0 under anaerobic incubation.Interestingly, further investigation of the anaerobic inhibition resulted in the discovery of a minor inactivation pathway involving covalent modification of flavin at a locus other than the isoalloxazine ring.Although the chemical nature of the new inhibitor-coenzyme adduct(s) has yet to be elucidated, a structure having MCPA-CoA linked to the N(10) ribityl side chain is appealing.The mechanistic insights derived from this study provide compelling evidence supporting our early notion that inactivation of MCAD by MCPA-CoA is likely to proceed through a radical mechanism.