Proton Abstraction Reaction, Steady-State Kinetics, and Oxidation−Reduction Potential of Human Glutaryl-CoA Dehydrogenase

Abstract

Glutaryl-CoA dehydrogenase catalyzes the oxidation of glutaryl-CoA to crotonyl-CoA and CO(2) in the mitochondrial degradation of lysine, hydroxylysine, and tryptophan. We have characterized the human enzyme that was expressed in Escherichia coli. Anaerobic reduction of the enzyme with sodium dithionite or substrate yields no detectable semiquinone; however, like other acyl-CoA dehydrogenases, the human enzyme stabilizes an anionic semiquinone upon reduction of the complex between the enzyme and 2,3-enoyl-CoA product. The flavin potential of the free enzyme determined by the xanthine-xanthine oxidase method is -0.132 V at pH 7.0, slightly more negative than that of related flavoprotein dehydrogenases. A single equivalent of substrate reduces 26% of the dehydrogenase flavin, suggesting that the redox equilibrium on the enzyme between substrate and product and oxidized and reduced flavin is not as favorable as that observed with other acyl-CoA dehydrogenases. This equilibrium is, however, similar to that observed in isovaleryl-CoA dehydrogenase. Comparison of steady-state kinetic constants of glutaryl-CoA dehydrogenase with glutaryl-CoA and the alternative substrates, pentanoyl-CoA and hexanoyl-CoA, suggests that the gamma-carboxyl group of glutaryl-CoA stabilizes the enzyme-substrate complex by at least 5.7 kJ/mol, perhaps by interaction with Arg94 or Ser98. Glu370 is positioned to function as the catalytic base, and previous studies indicate that the conjugate acid of Glu370 also protonates the transient crotonyl-CoA anion following decarboxylation [Gomes, B., Fendrich, G. , and Abeles, R. H. (1981) Biochemistry 20, 3154-3160]. Glu370Asp and Glu370Gln mutants of glutaryl-CoA dehydrogenase exhibit 7% and 0. 04% residual activity, respectively, with human electron-transfer flavoprotein; these mutations do not grossly affect the flavin redox potentials of the mutant enzymes. The reduced catalytic activities of these mutants can be attributed to reduced extent and rate of substrate deprotonation based on experiments with the nonoxidizable substrate analogue, 3-thiaglutaryl-CoA, and kinetic experiments. Determination of these fundamental properties of the human enzyme will serve as the basis for future studies of the decarboxylation reaction which is unique among the acyl-CoA dehydrogenases.

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

“…Previous structural and enzymatic studies of GCDH have repeatedly demonstrated FAD to be a tightly bound cofactor for this enzyme. Human GCDH recombinantly expressed and purified in another laboratory by methods similar to those used here revealed FAD bound in the crystal structure, although FAD had not been explicitly added at any stage (Dwyer et al, 2000;Goodman et al, 1995;Fu et al, 2004). Biochemical efforts to remove FAD from Pseudomonas fluorescens GCDH in another laboratory required multiple acid-ammonium precipitation steps, generating a very unstable protein which lost all activity if FAD was not added back within minutes (Gomes et al, 1981).…”

“…Unlike other flavoproteins within this family, GCDH (EC 1.3.99.7) performs , -dehydrogenation as well as decarboxylation of its substrate glutaryl-CoA to yield the product crotonyl-CoA (Dwyer et al, 2000). Transfer of electrons from the glutaryl-CoA precursor to the electron-transfer flavoprotein is mediated by the cofactor flavin adenine dinucleotide (FAD) in both prokaryotic and eukaryotic electron-transport chains (Tsai & Saier, 1995).…”

Decarboxylating and Nondecarboxylating Glutaryl-Coenzyme A Dehydrogenases in the Aromatic Metabolism of Obligately Anaerobic Bacteria

“…The human enzyme has been studied extensively, and a number of mutations in the gene coding for human GDH have been identified, which have been associated with cerebral aciduria type I (21). Meanwhile, details about the structure-function relationship and the catalytic mechanism have been elucidated in studies with human GDH (14,16,34,35). A conserved catalytic glutamate residue abstracts the ␣-proton from the substrate, which is followed by hydride transfer from the ␤-carbon to the flavin cofactor.…”

Identification of 3-Sulfinopropionyl Coenzyme A (CoA) Desulfinases within the Acyl-CoA Dehydrogenase Superfamily

“…2). This arginine was expected to be involved in binding of the sulfino group of 3SP-CoA (1), in analogy to glutaryl-CoA dehydrogenases, where an arginine residue is involved in binding of the terminal carboxyl group (44)(45)(46)(47)(48) prior to decarboxylation. Three residues (E87, S95, and Y369, according to the numbering for human glutaryl-CoA dehydrogenase) which probably enable the decarboxylating reaction are absent from nondecarboxylating glutaryl-CoA dehydrogenases and from all 3SP-CoA desulfinases.…”