title: Mechanism-based inactivation of human glutaryl-CoA dehydrogenase by 2-pentynoyl-CoA: rationale for enhanced reactivity.
abstract: 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.
authors.affiliation: Department of Pediatrics and Pharmaceutical Sciences, The University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: K Sudhindra
authors.initials: KS
authors.lastname: Rao
authors.forename: Mark
authors.initials: M
authors.lastname: Albro
authors.forename: Jerry
authors.initials: J
authors.lastname: Vockley
authors.forename: Frank E
authors.initials: FE
authors.lastname: Frerman
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D050770:Glutaryl-CoA Dehydrogenase; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000214:Acyl Coenzyme A; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C083148:2-pentynoyl-coenzyme A; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050770:Glutaryl-CoA Dehydrogenase