THE EFFECT OF AMINO-ACID SUBSTITUTION AT POSITION-219 OF CITROBACTER-FREUNDII CEPHALOSPORINASE ON EXTENSION OF ITS SUBSTRATE SPECTRUM

被引:13
作者
TSUKAMOTO, K [1 ]
OHNO, R [1 ]
NUKAGA, M [1 ]
SAWAI, T [1 ]
机构
[1] CHIBA UNIV,FAC PHARMACEUT SCI,DIV MICROBIAL CHEM,1-33 YAYOI CHO,CHIBA 263,JAPAN
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1992年 / 207卷 / 03期
关键词
D O I
10.1111/j.1432-1033.1992.tb17150.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 > Lys > Cys > Gln > 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.
引用
收藏
页码:1123 / 1127
页数:5
相关论文
共 25 条
  • [2] ANAN K, 1976, BASIC METHODS BIOCH, P113
  • [3] THE ACTIVE-SITE-SERINE PENICILLIN-RECOGNIZING ENZYMES AS MEMBERS OF THE STREPTOMYCES R61 DD-PEPTIDASE FAMILY
    JORIS, B
    GHUYSEN, JM
    DIVE, G
    RENARD, A
    DIDEBERG, O
    CHARLIER, P
    FRERE, JM
    KELLY, JA
    BOYINGTON, JC
    MOEWS, PC
    KNOX, JR
    [J]. BIOCHEMICAL JOURNAL, 1988, 250 (02) : 313 - 324
  • [4] TRANSFERABLE RESISTANCE TO CEFOTAXIME, CEFOXITIN, CEFAMANDOLE AND CEFUROXIME IN CLINICAL ISOLATES OF KLEBSIELLA-PNEUMONIAE AND SERRATIA-MARCESCENS
    KNOTHE, H
    SHAH, P
    KRCMERY, V
    ANTAL, M
    MITSUHASHI, S
    [J]. INFECTION, 1983, 11 (06) : 315 - 317
  • [5] THE ACYL-ENZYME MECHANISM OF BETA-LACTAMASE ACTION - THE EVIDENCE FOR CLASS-C BETA-LACTAMASES
    KNOTTHUNZIKER, V
    PETURSSON, S
    WALEY, SG
    JAURIN, B
    GRUNDSTROM, T
    [J]. BIOCHEMICAL JOURNAL, 1982, 207 (02) : 315 - 322
  • [6] LABIA R, 1988, REV INFECT DIS, V10, P885
  • [7] Miller J. H., 1972, EXPT MOL GENETICS, P433
  • [8] COVALENT BINDING OF MOXALACTAM TO CEPHALOSPORINASE OF CITROBACTER-FREUNDII
    MURAKAMI, K
    YOSHIDA, T
    [J]. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 1985, 27 (05) : 727 - 732
  • [9] MICRO-IODOMETRIC ASSAY FOR PENICILLINASE
    NOVICK, RP
    [J]. BIOCHEMICAL JOURNAL, 1962, 83 (02) : 236 - &
  • [10] REFINED CRYSTAL-STRUCTURE OF BETA-LACTAMASE FROM CITROBACTER-FREUNDII INDICATES A MECHANISM FOR BETA-LACTAM HYDROLYSIS
    OEFNER, C
    DARCY, A
    DALY, JJ
    GUBERNATOR, K
    CHARNAS, RL
    HEINZE, I
    HUBSCHWERLEN, C
    WINKLER, FK
    [J]. NATURE, 1990, 343 (6255) : 284 - 288