Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila

Abstract

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2'-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.

Kinetic study of penicillin acylase from Alcaligenes faecalis

“…A method based on the extremely effective stoichiometric inactivation by phenylmethylsulfonyl fluoride was earlier developed for active site titration of penicillin acylase from E. coli [26]. This method was later used for determination of active enzyme concentrations in highly and partially purified preparations of penicillin acylases from different strains of E. coli, Bacillus megaterium and Kluyvera citrophila [30–32]but was not effective for active site titration of penicillin acylases from Streptoverticillium sp. [33]and Xanthomonas sp.…”

High‐level Production, Chemical Modification and Site‐directed Mutagenesis of a Cephalosporin C Acylase from Pseudomonas Strain N176

“…Although it was difficult to identify the residue(s), we propose that the most possible candidate may be Ser239, the amino-terminal residue of the p chain, This was proposed from the following data: (a) the inactivation was independent of the carbamoylation of E amino groups of Lys residues, because sitedirected point mutants of each of 10 Lys residues to Gln residues retained acylase activity (Table 3); (b) the a amino group of the amino-terminal residue of either a or p chain of the inactivated acylase was blocked ; (c) the native amino-terminal sequence of the a chain appears non-essential for acylase activity according to data from amino-terminal variants (Table 1); (d) a mutant, [S239C]acylase, in which Ser239 was converted to Cys, showed dramatically reduced glutarylcephalosporanic acid acylase activity (approximately 1 % reduction) as compared to that of the wild-type enzyme, and its cephalosporin C acylase activity could not be detected (Table 3). Ser239 is perfectly conserved with other cephalosporin and penicillin G acylases (Ishii et al, 1994a, b) and corresponds to Ser290 (numbering corresponds to that of Barber0 et al, 1986) of penicillin G acylase which acts as a nucleophile in the catalysis Martin et al, 1991). Further investigation of the cephalosporin C acylase will be necessary to determine if Ser239 is an active residue for catalysis.…”

Structure, Processing and Catalytic Action of Penicillin Acylase

“…Recent results identify the N-terminal serine residue as a candidate for the active site nucleophile (Martin et al, 1990b;Slade etal., 1990).…”