Binding, hydration, and decarboxylation of the reaction intermediate glutaconyl-coenzyme A by human glutaryl-CoA dehydrogenase

Glutaconyl-coenzyme A (CoA) is the presumed enzyme-bound intermediate in th e oxidative decarboxylation of glutaryl-CoA that is catalyzed by glutaryl-C oA dehydrogenase. We demonstrated glutaconyl-CoA bound to glutaryl-CoA dehy drogenase after anaerobic reduction of the dehydrogenase with glutaryl-CoA. Glutaryl-CoA dehydrogenase also has intrinsic enoyl-CoA hydratase activity , a property of other members of the acyl-CoA dehydrogenase family. The enz yme rapidly hydrates glutaconyl-CoA at pH 7.6 with a k(cat) of 2.7 s(-1). T he k(cat) in the overall oxidation-decarboxylation reaction at pH 7.6 is ab out 9 s(-1). The binding of glutaconyl-CoA was quantitatively assessed from the K-m in the hydratase reaction, 3 muM, and the K-i, 1.0 muM, as a compe titive inhibitor of the dehydrogenase. These values compare with K-m and K- i of 4.0 and 12.9 muM, respectively, for crotonyl-CoA. Glu370 is the genera l base catalyst in the dehydrogenase that abstracts an alpha -proton of the substrate to initiate the catalytic pathway. The mutant dehydrogenase, Glu 370Gln, is inactive in the dehydrogenation and the hydratase reactions. How ever, this mutant dehydrogenase decarboxylates glutaconyl-CoA to crotonyl-C oA without oxidation-reduction reactions of the dehydrogenase flavin. Addit ion of glutaconyl-CoA to this mutant dehydrogenase results in a rapid, tran sient increase in long-wavelength absorbance (lambda (max) approximate to 7 25 nm), and crotonyl-CoA is found as the sole product. We propose that this 725 nm-absorbing species is the delocalized crotonyl-CoA anion that follow s decarboxylation and that the decay is the result of slow protonation of t he anion in the absence of the general acid catalyst, Glu370(H+). In the ab sence of detectable oxidation-reduction, the data indicate that oxidation-r eduction of the dehydrogenase flavin is not essential for decarboxylation o f glutaconyl-CoA.