3-Methyleneoctanoyl-CoA and 3-methyl-trans-2-octenoyl-CoA: two new mechanism-based inhibitors of medium chain acyl-CoA dehydrogenase from pig kidney.

The medium chain acyl-CoA dehydrogenase catalyzes the FAD-dependent oxidation of a variety of acyl-CoA substrates to the corresponding trans-2-enoyl-CoA thioesters. This work identifies 3-methyleneoctanoyl-CoA and 3-methyl-trans-2-octenoyl-CoA as representatives of a new class of mechanism-based inhibitor of the dehydrogenase. One equivalent of either compound generates an inactive reduced flavin species with low absorption at 450 nm and a shoulder at 320 nm suggestive of an N-5 adduct. Reduction is rapid with the 3-methylene analogue (10/s at 1 degree C), but comparatively slow for 3-methyl-trans-2-octenoyl-CoA (1.1 x 10(-4)/s, under the same conditions). The reduced species is very stable, but the adduct can be slowly displaced with a large excess of octanoyl-CoA. The reduced adduct resists oxidation by the facile one-electron oxidant of the dehydrogenase, ferricenium hexafluorophosphate. Evidence that both isomeric inhibitors generate the same reduced flavin species includes an essentially identical visible spectrum, the same kinetics of displacement using octanoyl-CoA, and the same mixture of products on HPLC after denaturation of the treated enzyme with trichloroacetic acid, methanol, or by boiling. Experiments with the corresponding shorter analogues of these inhibitors, 3-methylenebutanoyl-CoA and 3-methyl-2-butenoyl-CoA confirm and extend these findings. These reduced adducts are less stable, allowing the dehydrogenase to catalyze the interconversion of the unconjugated 3-methylenebutanoyl-CoA to the more stable conjugated 3-methyl-2-butenoyl-CoA thioester (Keq ca. 150). These data suggest that alpha-proton abstraction from the 3-methylene derivatives or gamma-proton removal from the 3-methyl-2-enoyl analogues generates a common carbanionic intermediate which attacks oxidized flavin. As would be expected, the unconjugated 3-methylene derivatives are more effective inhibitors of the dehydrogenase than the thermodynamically more stable 3-methylenoyl analogues.