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The incorporation of nitrotyrosine into 3-phosphoglycerate kinase activated by carbodiimide results in the chemical modification of a single essential residue. After total proteolytic digestion, isolation of the dipeptide gamma Glu-NO2 Tyr by immuno-affinity chromatography indicates the implication of a glutamyl residue. It is interesting to point out the applicability of the method described for the purification of peptides containing carboxyl residues.

3-Phosphoglycerate kinase is inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate and nitrotyrosine ethyl ester. The coupling of 1 mol nitrotyrosine/mol enzyme is sufficient to inactivate the protein completely. A weak protection against inactivation is observed with each substrate added separately. In contrast, the complex ATP--3-phosphoglycerate--enzyme or ATP--Mg--3-phosphoglycerate--enzyme affords a considerable protection. The critical residue is identified as a glutamyl residue after isolation by immuno-affinity chromatography of nitrotyrosyl peptide resulting from exhaustive proteolytic digestion of the modified protein. In addition, the determination of the primary sequence of the C-terminal part of the protein leads to the location of the glutamyl residue at position eight from the C-terminus. We conclude that this glutamyl residue is situated in the domain which does not bind the nucleotide substrates [Bryant, T.N., Watson, H.C. and Wendell, P.L. (1974) Nature (Lond.) 247, 14--17]. Its role in the catalysis process is discussed.