Mechanism-based inactivation of human glutaryl-CoA dehydrogenase by 2-pentynoyl-CoA: rationale for enhanced reactivity

2-Pentynoyl-CoA inactivates glutaryl-CoA dehydrogenase at a rate that considerably exceeds the rates of inactivation of short chain and medium chain acyl-CoA dehydrogenases by this inhibitor and related 2-alkynoyl-CoAs. To determine the rate of inactivation by 2-pentynoyl-CoA, we investigated the inactivation in the presence of a non-oxidizable analog, 3-thiaglutaryl-CoA, which competes for the binding site. The enhanced rate of inactivation does not reflect an alteration in specificity for the acyl group, nor does it reflect the covalent modification of a residue other than the active site glutamate. In addition to determining the inactivation of catalytic activity a spectral intermediate was detected by stopped-flow spectrophotometry, and the rate constants of formation and decay of this charge transfer complex (lambdamax approximately 790 nm) were determined by global analysis. Although the rate-limiting step in the inactivation of the other acyl-CoA dehydrogenases can involve the abstraction of a proton at C-4, this is not the case with glutaryl-CoA dehydrogenase. Glutaryl-CoA dehydrogenase is also differentiated from other acyl-CoA dehydrogenases in that the catalytic base must access both C-2 and C-4 in the normal catalytic pathway. Access to C-4 is not obligatory for the other dehydrogenases. Analysis of the distance from the closest carboxylate oxygen of the glutamate base catalyst to C-4 of a bound acyl-CoA ligand for medium chain, short chain, and isovaleryl-CoA dehydrogenases suggests that the increased rate of inactivation reflects the carboxylate oxygen to ligand C-4 distance in the binary complexes. This distance for wild type glutaryl-CoA dehydrogenase is not known. Comparison of the rate constants of inactivation and formation of a spectral species between wild type glutaryl-CoA dehydrogenase and a E370D mutant are consistent with the idea that this distance in glutaryl-CoA dehydrogenase contributes to the enhanced rate of inactivation and the 1,3-prototropic shift catalyzed by the enzyme.