Studies of glutamate dehydrogenase. Methionine-169: the preferentially carboxymethylated residue.
In phosphate buffer at pH 7.0, 5,5'-dithio-bis(2-nitrobenzoic acid), N-ethylmaleimide or iodoacetamide do not alter the activity of beef liver glutamate dehydrogenase. Iodoacetate, however, inactivities the enzyme irreversibility by alkylation. Combined addition of the coenzyme NADH and the substrate 2-oxoglutarate or the effector GTP protects against this inactivation. The alkylation reaction is independent of pH between pH 6-9 indicating that amino, imidazole or phenolic groups are probably not involved in this reaction. Titration of the thiol groups, after denaturation of the enzyme, revealed the loss of approximately one group per polypeptide chain. However, this is not due to the exclusive alkylation of a cysteine residue, since alkylation with iodo-[2-14C]acetic acid also labels a methionine residue. 50% of the label is incorporated into methionine-169 and only 7% into cysteine-115, the remaining radioactivity is distributed in minor quantities (4%) in several unidentified residues. A probable cause of the erroneous thiol groups titration is discussed.
Studies of glutamate dehydrogenase. Methionine-169: the preferentially carboxymethylated residue. In phosphate buffer at pH 7.0, 5,5'-dithio-bis(2-nitrobenzoic acid), N-ethylmaleimide or iodoacetamide do not alter the activity of beef liver glutamate dehydrogenase. Iodoacetate, however, inactivities the enzyme irreversibility by alkylation. Combined addition of the coenzyme NADH and the substrate 2-oxoglutarate or the effector GTP protects against this inactivation. The alkylation reaction is independent of pH between pH 6-9 indicating that amino, imidazole or phenolic groups are probably not involved in this reaction. Titration of the thiol groups, after denaturation of the enzyme, revealed the loss of approximately one group per polypeptide chain. However, this is not due to the exclusive alkylation of a cysteine residue, since alkylation with iodo-[2-14C]acetic acid also labels a methionine residue. 50% of the label is incorporated into methionine-169 and only 7% into cysteine-115, the remaining radioactivity is distributed in minor quantities (4%) in several unidentified residues. A probable cause of the erroneous thiol groups titration is discussed.