title: Characterization of the general anion-binding site in glutamate dehydrogenase-NADPH complex.
abstract: The reductive amination of alpha-ketoglutarate, catalyzed by bovine liver glutamate dehydrogenase, is inhibited by various anions. Formate and acetate ions are competitive with alpha-ketoglutarate. The pH dependence of the pKi profiles for these anions reveals that they bind to the enzyme-NADPH complex only when an enzymatic residue of pK 8.0 +/- 0.1 in the binary complex is protonated. The ionization of this residue has a delta Hion of 15 +/- 4 kcal/mol. These pK and delta Hion values are not significantly different from those observed in the same complex for the enzyme group which binds the gamma-CO2- of alpha-ketoglutarate and oxalylglycine. It is concluded that formate and acetate also bind to the gamma-carboxylate site in enzyme-NADPH. The Ki values for formate and acetate in a buffer containing 0.1 M phosphate are 20 +/- 4 and 32 +/- 5 mM, respectively, when the pK 8.0 group is fully protonated. Phosphate and trifluoroacetate also show an inhibitory effect, while valerate and sulfate have little effect on the reductive amination rates. The results suggest that specific anions can bind to the gamma-carboxylate site by ionic interactions and alter the kinetic and thermodynamic parameters of the glutamate dehydrogenase-NADPH complex in significant ways.
authors.affiliation: Department of Chemistry/Physics, Uniersity of Alaska, Anchorage 99508.
authors.forename: R
authors.initials: R
authors.lastname: Srinivasan
mesh_terms: D000085:Acetates / Q000494:pharmacology; D000586:Amination; D000818:Animals; D000838:Anions; D001665:Binding Sites; D002417:Cattle; D005561:Formates / Q000494:pharmacology; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007656:Ketoglutaric Acids / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D000085:Acetates; D000838:Anions; D005561:Formates; D007656:Ketoglutaric Acids; D009249:NADP; D005969:Glutamate Dehydrogenase