title: The effect of amino acid substitution at position 219 of Citrobacter freundii cephalosporinase on extension of its substrate spectrum.
abstract: The cephalosporinase of Citrobacter freundii GN346 is a class-C beta-lactamase comprising 361 amino acids. The substitution of the glutamic acid at position 219 in the enzyme by lysine was previously shown to broaden its substrate specificity to unfavorable substrates such as oxyimino cephalosporins [Tsukamoto, K., Ohno, R. & Sawai, T. (1990) J. Bacteriol. 172, 4348-4351]. To investigate the cause of this phenomenon, Glu219 was changed to glutamine, cysteine or tryptophan. All the resultant enzymes showed higher cefuroxime-hydrolytic activities than the wild type, the order of increasing cefuroxime-hydrolytic activity being as follows: Trp greater than Lys greater than Cys greater than Gln greater than Glu. The rate of hydrolysis of cefuroxime by the Trp219 enzyme was approximately 3 x 10(4) times that of the wild-type enzyme. The order of increasing cefuroxime hydrolysis was approximately proportional to the molecular volume of the amino acid substituted and independent of the ionic character of the amino acids. The cysteine residue at position 219 in the Cys219 enzyme allowed its complete reaction with an SH-blocking reagent, 4-chloromercuriphenylsulfonic acid. The modified enzyme with the bulkier residue showed a 45% higher cefuroxime-hydrolytic activity than the untreated enzyme. These results suggested that extension of the substrate spectrum may be attributed to alteration in the configuration of the enzyme around position 219.
authors.affiliation: Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Tsukamoto
authors.forename: R
authors.initials: R
authors.lastname: Ohno
authors.forename: M
authors.initials: M
authors.lastname: Nukaga
authors.forename: T
authors.initials: T
authors.lastname: Sawai
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D001483:Base Sequence; D002510:Cephalosporinase / Q000235:genetics* / Q000378:metabolism; D016971:Citrobacter freundii / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D013379:Substrate Specificity; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D000596:Amino Acids; D065093:beta-Lactamase Inhibitors; D002510:Cephalosporinase; D001618:beta-Lactamases