title: The function of Arg-94 in the oxidation and decarboxylation of glutaryl-CoA by human glutaryl-CoA dehydrogenase.
abstract: Glutaryl-CoA dehydrogenase catalyzes the oxidation and decarboxylation of glutaryl-CoA to crotonyl-CoA and CO(2). 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 gamma-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 k(cat) of the mutant dehydrogenases to 2-3% of k(cat) of the wild type enzyme. K(m) 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 alpha-proton from 3-thiaglutaryl-CoA, to yield a charge transfer complex with the oxidized flavin, is severely limited. In contrast, abstraction of the alpha-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 alpha-proton of glutaryl-CoA and 3-thiaglutaryl-CoA, both of which contain gamma-carboxylates. It is also possible that Arg-94 may orient glutaryl-CoA and 3-thiaglutaryl-CoA for abstraction of an alpha-proton.
authors.affiliation: Department of Pediatrics, The University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: T M
authors.initials: TM
authors.lastname: Dwyer
authors.forename: K S
authors.initials: KS
authors.lastname: Rao
authors.forename: J B
authors.initials: JB
authors.lastname: Westover
authors.forename: J J
authors.initials: JJ
authors.lastname: Kim
authors.forename: F E
authors.initials: FE
authors.lastname: Frerman
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D005977:Glutarates / Q000378:metabolism; D050770:Glutaryl-CoA Dehydrogenase; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D005977:Glutarates; D011994:Recombinant Proteins; C010667:acetoacetyl CoA; C015901:glutaryl-coenzyme A; C044181:hexanoyl-coenzyme A; D001120:Arginine; C010701:crotonyl-coenzyme A; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050770:Glutaryl-CoA Dehydrogenase