Methionyl Residue Critical for Activity and Regulation of Bovine Liver Glutamate Dehydrogenase

Abstract

Abstract Glutamate dehydrogenase is inactivated by incubation with 4-iodoacetamidosalicylic acid at 37° and pH 6.0. The reaction obeys pseudo-first order kinetics, with a rate constant which is linearly proportional to the 4-iodoacetamidosalicylic acid concentration. A 4- to 5-fold decrease in the rate of inactivation is provided by the substrates α-ketoglutarate and glutamate when added to the incubation mixture with ADP and DPN, but no appreciable decrease in the inactivation rate is produced by the coenzymes themselves, either alone or when combined with ADP or GTP. These results suggest that 4-iodoacetamidosalicylic acid reacts in the region of the active site. 4-Iodoacetamidosalicylic acid produces a change in the response of the enzyme to the allosteric inhibitor GTP, as exemplified by an increase in the kinetic dissociation constant of the enzyme-coenzyme-GTP complex. Although the reagent causes inactivation of the enzyme in the absence of GTP, it does not influence the small residual activity (4 to 8%) seen when a saturating concentration of GTP is present. Only methionyl and cysteinyl residues of glutamate dehydrogenase are altered by 4-iodoacetamidosalicylic acid at periods up to 10 times the half-life for inactivation; lysyl and histidyl residues are not alkylated during this time. As determined by the release of iodide ion from 4-iodoacetamidosalicylic acid, approximately 4 amino acids per peptide chain are modified when the enzyme loses 90% of its activity: about 2 cysteinyl and 2 methionyl residues are alkylated. Several lines of evidence suggest that covalent modification of cysteine is not the primary cause of inactivation. The rate of alkylation of sulfhydryl groups (K-sh = 0.0015 min-1) is only one-sixth that of inactivation (Ki = 0.0102 min-1) for the enzyme in the absence of ligands. These two rate constants can be varied independently by the addition of substrates and allosteric modifiers. In contrast, a good correlation is observed between the degree of inactivation of the enzyme and the extent of modification of approximately 1 methionyl residue per peptide chain. Furthermore, treatment of 75% inactivated enzyme with dithiothreitol produces a 3-fold increase in the enzymatic activity, accompanied by the regeneration of methionine but no appreciable alteration of the number of modified cysteine residues. It is concluded that the integrity of a methionine residue is critical for the function of glutamate dehydrogenase.