title: Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105. Effect on conformation and catalytic activity.
abstract: Penicillin acylase (EC 3.5.1.11) was completely inactivated with equimolar phenylmethane [35S]sulphonyl fluoride (PhMe35SO2F); the stability of the sulphonyl group in the modified protein was determined by measurement of the radioactivity in ultrafiltrates. In 8 M urea, the rate of loss of the sulphonyl group was similar to that observed in PhMeSO2F-inactivated chymotrypsin [Gold, A.M. & Fahrney, D. (1964) Biochemistry 3, 783-791]. Incubation of the PhMeSO2F-inactivated acylase with 0.7 M potassium thioacetate yielded an acetylthiol enzyme which was subsequently converted to a thiol-enzyme during incubation with 10 mM 6-aminopenicillanic acid. 4-Pyridyl-ethylcysteine was released by acid hydrolysis after reaction of the thiol-protein with 4-vinylpyridine. The rates of reaction of thiol-penicillin acylase with iodoacetic acid and 2,2'-dipyridyl disulphide were consistent with the presence of an incompletely accessible cysteinyl sidechain. After carboxymethylating the thiol-enzyme with iodo[2-3H]acetic acid, the label was shown by SDS/PAGE and sequencing analysis to be associated exclusively with the beta-chain NH2-terminal residue, indicating conversion of Ser290 to S-carboxymethyl-cysteine. Near-ultraviolet CD spectra showed the conformation of thiol-penicillin acylase to be indistinguishable from that of the native protein but the catalytic activity was less than 0.02% of that of the normal enzyme. The possibility that Ser290 acts as a nucleophile in catalysis is discussed.
authors.affiliation: Department of Biochemistry and Genetics, School of Biomedical and Biomolecular Sciences, University of Newcastle upon Tyne, England.
authors.forename: A
authors.initials: A
authors.lastname: Slade
authors.forename: A J
authors.initials: AJ
authors.lastname: Horrocks
authors.forename: C D
authors.initials: CD
authors.lastname: Lindsay
authors.forename: B
authors.initials: B
authors.lastname: Dunbar
authors.forename: R
authors.initials: R
authors.lastname: Virden
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010397:Penicillanic Acid / Q000494:pharmacology / Q000031:analogs & derivatives; D010399:Penicillin Amidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D011725:Pyridines / Q000494:pharmacology; D012694:Serine; D013462:Sulfur Radioisotopes
chemical_list: D011725:Pyridines; D013462:Sulfur Radioisotopes; D012694:Serine; D010664:Phenylmethylsulfonyl Fluoride; D010397:Penicillanic Acid; D010399:Penicillin Amidase; C029351:4-vinylpyridine; D003545:Cysteine; C004408:aminopenicillanic acid