Cephalosporin C (CC) acylase from Pseudomonas strain N176 was chemically modified by tetranitromethane (TNM), causing complete loss of activity. Modification using molar excesses of TNM up to 10 resulted in complete inactivation when 1.4 mol tyrosines/mol enzyme were modified. Digestion of native and TNM-modified acylase with Achromobacter protease I (API), separation by high performance liquid phase chromatography (HPLC) and amino terminal sequencing of the resultant peptides were used to identify the modified tyrosine residues, The major difference in HPLC profile between these API digests was shown to be the peak corresponding to the peptide Ser(239)-Lys(301) of native acylase. A portion of the peak for the peptide Ala(45)-Lys(73) was also shifted in HPLC analysis of TNM-modified acylase. The peptides isolated from the modified acylase were shown to contain nitrated tyrosines (3-nitrotyrosine) at positions 270 and 52, respectively. These findings indicate that Tyr(270) is completely modified, and Tyr(52) is partially modified in the inactivated acylase. Each of the fifteen tyrosines in the acylase was altered to leucine by site-directed mutagenesis to complement the chemical modification with TNM. At pH 8.7, the mutant acylase in which tyrosine at position 270 is changed to leucine showed GL-7ACA and CC acylase activities reduced to 28.0 and 32.2% of native acylase, respectively. The results correspond to those obtained from TNM-modification. A similar reduction in activity was also obtained in the case of Tyr(491) mutant, although nitration of this residue was not confirmed by chemical modification. Therefore Tyr(270) and Tyr(491) are important for exerting the maximum activity of the enzyme, but are not essential for catalysis. However, mutation of Tyr(52) to Leu produced little change in acylase activity. The mutant acylase in which Tyr(705) is changed to leucine has a lowered pH optimum for GL-7ACA, which may be useful for further improvement of the acylase.