Mechanism of action of glutaryl-CoA and butyryl-CoA dehydrogenases. Purification of glutaryl-CoA dehydrogenase.

Glutaryl-CoA dehydrogenase, a flavoprotein, catalyzes the reaction -OOCCH3CH2--CH2COSR (FAD leads to FADH2) leads to CH3CH = CHCOSR + CO2 (SR = CoA or pantetheine). With the isolated enzyme, a dye serves as the final electron acceptor. The enzyme from Pseudomonas fluorescens (ATCC 11250) has been purified to homogeneity. It was established with appropriate isotopic substitutions that the proton which is added to the gamma position of the product, subsequent to decarboxylation, is not derived from the solvent but is derived from the alpha position of the substrate. Under conditions where no net conversion of substrate occurs, i.e., in the absence of electron acceptor, the enzyme catalyzes the exchange of the beta hydrogen of the substrate with solvent protons. Butyryl-CoA dehydrogenase (M. elsedenii), which catalyzes an analogous reaction, catalyzes the exchange of both the alpha and beta hydrogens with solvent protons in the absence of electron acceptor. Glutaryl-CoA dehydrogenase and butyryl-CoA dehydrogenase are irreversibly inactivated by the substrate analogues 3-butynoylpantetheine and 3-pentynoylpantetheine. These inactivators do not form an adduct with the flavin and probably react with a nucleophile at the active site. Upon inactivation, the spectrum of the enzyme-bound flavin is essentially unchanged, and the flavin can be reduced by Na2S2O4. We suggest that inactivation involves intermediate allene formation. We proposed that these results support an oxidation mechanism for glutaryl-CoA dehydrogenase and butyryl-CoA dehydrogenase which is initiated by proton abstraction. With glutaryl-CoA dehydrogenase, the base, which abstracts the substrate alpha proton, is shielded from the solvent and is then used to protonate the carbanion (CH2--CH--CHCOSCoA) formed after oxidation and decarboxylation.