Studies of Glutamate Dehydrogenase

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Abstract
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 inactivates the enzyme irreversibly 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 1 group/polypeptide chain. 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. Part of the label (50%) is incorporated into Met-169 and only 7% into Cys-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.