NADP+-specific glutamate dehydrogenase from Salmonella typhimurium, cloned and expressed in Escherichia coli, has been purified to homogeneity. The nucleotide sequence of S. typhimurium gdhA was determined and the amino acid sequence derived. The nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) reacts irreversibly with the enzyme to yield a partially inactive enzyme. After about 60% loss of activity, no further inactivation is observed. The rate of inactivation exhibits a nonlinear dependence on 2-BDB-T epsilon A-2',5'-DP concentration with kmax = 0.160 min-1 and KI = 300 microM. Reaction of 200 microM 2-BDB-T epsilon A-2',5'-DP with glutamate dehydrogenase for 120 min results in the incorporation of 0.94 mol of reagent/mol of enzyme subunit. The coenzymes, NADPH and NADP+, completely protect the enzyme against inactivation by the reagent and decrease the reagent incorporation from 0.94 to 0.5 mol of reagent/mol enzyme subunit, while the substrate alpha-ketoglutarate offers only partial protection. These results indicate that 2-BDB-T epsilon A-2',5'-DP functions as an affinity label of the coenzyme binding site and that specific reaction occurs at only about 0.5 sites/enzyme subunit or 3 sites/hexamer. Glutamate dehydrogenase modified with 200 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of coenzyme was reduced with NaB3H4, carboxymethylated, and digested with trypsin. Labeled peptides were purified by high performance liquid chromatography and characterized by gas phase sequencing. Two peptides modified by the reagent were isolated and identified as follows: Phe-Cys(CM)-Gln-Ala-Leu-Met-Thr-Glu-Leu-Tyr-Arg and Leu-Cys(CM)-Glu-Ile-Lys. These two peptides were located within the derived amino acid sequence as residues 146-156 and 282-286. In the presence of NADPH, which completely prevents inactivation, only peptide 146-156 was labeled. This result indicates that modification of the pentapeptide causes loss of activity. Glutamate 284 in this peptide is the probable reaction target and is located within the coenzyme binding site.