title: 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.
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Martín
authors.forename: A
authors.initials: A
authors.lastname: Slade
authors.forename: A
authors.initials: A
authors.lastname: Aitken
authors.forename: R
authors.initials: R
authors.lastname: Arche
authors.forename: R
authors.initials: R
authors.lastname: Virden
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004755:Enterobacteriaceae / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010399:Penicillin Amidase / Q000737:chemistry* / Q000187:drug effects; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D012694:Serine / Q000031:analogs & derivatives*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology
chemical_list: D013438:Sulfhydryl Compounds; D012694:Serine; D010664:Phenylmethylsulfonyl Fluoride; D010399:Penicillin Amidase; C005732:thioacetic acid