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 g lutaryl-CoA to crotonyl-CoA and CO2. Inherited defects in the protein cause glutaric acidemia type I, a fatal neurologic disease. Glutaryl-CoA dehydro genase is the only member of the acyl-CoA dehydrogenase family with a catio nic residue, Arg-94, situated in the binding site of the acyl moiety of the substrate. Crystallographic investigations suggest that Argc94 is within h ydrogen bonding distance of the gamma -carboxylate of glutaryl-CoA Substitu tion of Arg-94 by glycine, a disease-causing mutation, and by glutamine, wh ich is sterically more closely related to arginine, reduced K-cat of the mu tant dehydrogenases to 2-3% of k(ca)t of the wild type enzyme. K-m of these mutant dehydrogenases for glutaryl-CoA increases 10- to 16-fold. The stead y-state kinetic constants of alternative substrates, hexanoyl-CoA and gluta ramyl-CoA, which are not decarboxylated, are modestly affected by the mutat ions. The latter changes are probably due to steric and polar effects. The dissociation constants of the non-oxidizable substrate analogs, 3-thiagluta ryl-CoA and acetoacetyl-CoA, are not altered by the mutations. However, abs traction of a cy-proton from 3-thiaglutaryl-CoA, to yield a charge transfer complex with the oxidized flavin, is severely limited. In contrast, abstra ction of the alpha -proton of acetoacetyl-CoA by Arg-94 --> Gin mutant dehy drogenase is unaffected, and the resulting enolate forms a charge transfer complex with the oxidized flavin, These experiments indicate that Arg-94 do es not make a major contribution to glutaryl-CoA binding. However, the elec tric field of Arg-94 may stabilize the dianions resulting from abstraction of the alpha -proton of glutaryl-CoA and 3-thiaglutaryl-CoA, both of which contain gamma -carbaxylates. It is also possible that Arg-94 may orient glu taryl-CoA and 3-thiaglutaryl-CoA for abstraction of an alpha -proton.