Affinity Labelling of Cephalosporin C Acylase from Pseudomonas sp. : N176 with a Substrate Analogue, 7β-(6-Bromohex-anoylamido)cephalosporanic Acid

We synthesized 7β-(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 strainght line (k_<max>=0.113 min^<-1>, K_1=0.51 mM). 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 6BH-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 α-chymotrptic digestion followed by amino acid sequence analysis, Tyr^<270> was determined as the site labelled by 6-BH-7ACA. Replacing Try^<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.