Open AccessThe effect of amino acid substitution at position 219 of Citrobacter freundii cephalosporinase on extension of its substrate spectrum
Author(s) -
TSUKAMOTO Kikuo,
OHNO Reiko,
NUKAGA Michiyoshi,
SAWAI Tetsuo
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb17150.x
Subject(s) - citrobacter freundii , chemistry , hydrolysis , stereochemistry , amino acid , enzyme , cysteine , tryptophan , active site , biochemistry , klebsiella pneumoniae , escherichia coli , gene
The cephalosporinase of Citrobacter freundii GN346 is a class‐C β‐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 subrate 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 follow: Trp > Lys > Cys > Gln > Glu. The rate of hydrolysis of cefuroxime by the Trp219 enzyme was approximately 3×10 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 acid. 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.