Effect of Carboxyl Group Modification on Some of the Enzymatic Properties of L-Glutamate Dehydrogenase

• Published in: Journal of biochemistry (Tokyo) 1973-07, Vol.74 (1), p.77-86
• Main Authors: SWAISGOOD, Harold, NATAKE, Masato
• Format: Article
• Language: English
• Subjects: Animals; Carbodiimides; Cattle; Chemical Phenomena; Chemistry; Electrophoresis; Glutamate Dehydrogenase; Glycine; Kinetics; Liver - enzymology; NAD; Oxidation-Reduction
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a130233

The carboxyl groups of bovine liver L-glutamate dehydrogenase [EC 1.4.1.3] were modified to various extents by coupling with glycine methyl ester in the presence of l-ethyl-3-dimethylaminopropylcarbodiimide (EDC). By properly selecting the reaction conditions, the number of carboxyl groups modified per polypeptide chain could be varied from 2–3 to nearly complete modification. In. the ‘ native‘ form, 30–40 fewer carboxyl groups react with EDC than under conditions favoring unfolding and subunit dissociation. Comparison of the number reacting with the theoretical number available (1,2) shows that essentially all groups are reactive under the latter conditions. The less reactive groups may participate in tyrosyl-carboxylate hydrogen-bonds at the site of subunit interactions [3, 4). Kinetic studies of the enzymatic oxidation of NADH or reduction of NAD+ by two modified enzyme preparations indicated a general increase in their apparent Michaelis constants as compared to those of the native enzyme. The modified enzymes, however, showed greater degrees of activation by high glutamate concentrations.. Moreover, the modified enzymes lost all of the substrate inhibition effects displayed by the native enzyme for NADH and α-ketoglutarate.