The reactivity of a functional tyrosyl residue in carboxypeptidase B. Nitration of the cadmium enzyme.

Cadmium-carboxypeptidase B was nitrated with tetranitromethane. The enzyme polymerized extensively during nitration. In the monomer nitrated Cd-carboxypeptidase B, 70% of the activity of Cd-carboxypeptidase B was retained. In order to identify the tyrosyl residues nitrated, the enzyme was digested with chymotrypsin and subtilisin and the nitrotyrosyl peptides were purified by affinity chromatography on antityrosyl-antibody-Sepharose conjugate followed by two-dimensional thin-layer chromatography. The major nitropeptides, representing 65% of the nitrotyrosyl label, were compatible with the segment of the sequence containing Tyr-240 and Tyr-259. Only 10% of the nitrotyrosyl label was found in the segment of Tyr-248. This indicates that the state of Tyr-248 in Cd-carboxypeptidase B differs from that in zinc-carboxypeptidase B where it shows chemical hyperreactivity due to its proximity to the metal ion.