The Function of Arg-94 in the Oxidation and Decarboxylation of Glutaryl-CoA by Human Glutaryl-CoA Dehydrogenase

Glutaryl-CoA dehydrogenase catalyzes the oxidation and decarboxylation of glutaryl-CoA to crotonyl-CoA and CO2. Inherited defects in the protein cause glutaric acidemia type I, a fatal neurologic disease. Glutaryl-CoA dehydrogenase is the only member of the acyl-CoA dehydrogenase family with a cationic residue, Arg-94, situated in the binding site of the acyl moiety of the substrate. Crystallographic investigations suggest that Arg-94 is within hydrogen bonding distance of the γ-carboxylate of glutaryl-CoA. Substitution of Arg-94 by glycine, a disease-causing mutation, and by glutamine, which is sterically more closely related to arginine, reduced kcat of the mutant dehydrogenases to 2–3% of kcat of the wild type enzyme. Km of these mutant dehydrogenases for glutaryl-CoA increases 10- to 16-fold. The steady-state kinetic constants of alternative substrates, hexanoyl-CoA and glutaramyl-CoA, which are not decarboxylated, are modestly affected by the mutations. The latter changes are probably due to steric and polar effects. The dissociation constants of the non-oxidizable substrate analogs, 3-thiaglutaryl-CoA and acetoacetyl-CoA, are not altered by the mutations. However, abstraction of a α-proton from 3-thiaglutaryl-CoA, to yield a charge transfer complex with the oxidized flavin, is severely limited. In contrast, abstraction of the α-proton of acetoacetyl-CoA by Arg-94 → Gln mutant dehydrogenase is unaffected, and the resulting enolate forms a charge transfer complex with the oxidized flavin. These experiments indicate that Arg-94 does not make a major contribution to glutaryl-CoA binding. However, the electric field of Arg-94 may stabilize the dianions resulting from abstraction of the α-proton of glutaryl-CoA and 3-thiaglutaryl-CoA, both of which contain γ-carboxylates. It is also possible that Arg-94 may orient glutaryl-CoA and 3-thiaglutaryl-CoA for abstraction of an α-proton.