Functional arginine residues involved in coenzyme binding by glutamate dehydrogenases.
Reaction of the NADP-dependent glutamate dehydrogenase of Neurospora with 1,2-cyclohexanedione results in a biphasic loss of enzyme activity. At the end of the rapid phase of the reaction (t1/2 = 1.5 min) the enzyme activity is diminished by approximately 60% with the simultaneous loss of 1 residue of arginine per subunit. After 60 min, the enzyme activity is completely lost with the modification of a total of 2 arginine residues per subunit. Reaction of bovine liver glutamate dehydrogenase with cyclohexanedione causes a rapid loss of approximately 45% of the enzyme activity and modification of about 1.5 residues of arginine per subunit. More prolonged treatment results in reaction of an additional 4 residues of arginine per subunit but is without further effect on the residual activity. The activity of the Neurospora enzyme is not protected by substrate, coenzyme, or a combination of both; however, the activity of the bovine enzyme is partially protected by high levels of NAD or NADP. Although the Km for alpha-ketoglutarate is unchanged by a limited modification of either enzyme with cyclohexanedione, the Km for coenzyme is increased about 2-fold for the Neurospora enzyme and about 1.5-fold for the bovine enzyme. The Ki of the Neurospora dehydrogenase for the competitive inhibitor 2'-monophosphoadenosine-5'-diphosphoribose is unchanged by the enzyme modification, but nicotinamide mononucleotide, a competitive inhibitor for the native Neurospora enzyme, does not inhibit the glutamate dehydrogenase with 1 modified arginine residue. This finding implies that the modified arginine is at or near the nicotinamide binding iste of the enzyme.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001120:Arginine / Q000032:analysis*; D001665:Binding Sites; D002417:Cattle; D003512:Cyclohexanones; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009243:NAD; D009491:Neurospora / Q000201:enzymology; D011485:Protein Binding; D013045:Species Specificity
D000596:Amino Acids; D003512:Cyclohexanones; D009243:NAD; D001120:Arginine; D005969:Glutamate Dehydrogenase