We synthesized 7 beta(6-bromohexanoylamido)cephalosporanic acid (6-BH-7ACA), a substrate analogue of an acylase from Pseudomonas N176 (N176 acylase) to determine the substrate binding site of the acylase. The enzyme was inactivated by incubation with 6-BH-7ACA in a time-dependent manner. A double reciprocal plot of the pseudo-first-order rate constant (k(obs)) against the 6-BH-7ACA concentration gave a straight line (k(max)=0.113 min(-1), K-I=0.51mM). A plot of log k(obs) against log [6-BH-7ACA] was linear with a slope of 0.87. Inactivation of the enzyme with 6-BH-7ACA was inhibited by addition of 7-aminocephalosporanic acid and glutaric acid. These data indicate that 6-BH-7ACA functions as an affinity label reagent and the inactivation by 6-BH-7ACA is due to the modification of a single residue located in the neighborhood of the substrate binding region of the acylase. The digest of the inactivated enzyme with lysylendopeptidase was fractionated by reversed phase high-performance liquid chromatography (HPLC). One fragment was eluted with a different retention time from the corresponding fragment of the intact enzyme. From additional alpha-chymotryptic digestion followed by amino acid sequence analysis, Tyr(270) was determined as the site labelled by 6-BH-7ACA. Replacing Tyr(270) with a Phe residue by site-directed mutagenesis caused a decrease in the enzyme capability (k(cat)/K-m). While the K-m of the mutant acylase increased slightly, the k(cat) decreased to about 50% of that of the wild-type. These results indicate that although labelled Tyr(270) is not essential, it does play an important role in the enzymatic activity.