SITE-DIRECTED MUTAGENESIS OF BETA-LACTAMASE-I - SINGLE AND DOUBLE MUTANTS OF GLU-166 AND LYS-73

被引:131
作者
GIBSON, RM
CHRISTENSEN, H
WALEY, SG
机构
[1] UNIV OXFORD, SIR WILLIAM DUNN SCH PATHOL, OXFORD OX1 3RE, ENGLAND
[2] OXFORD CTR MOLEC SCI, OXFORD 0X1 3QY, ENGLAND
关键词
D O I
10.1042/bj2720613
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
Two single mutants and the corresponding double mutant of beta-lactamase I from Bacillus cereus 569/H were constructed and their kinetics investigated. The mutants have Lys-73 replaced by arginine (K73R), or Glu-166 replaced by aspartic acid (E166D), or both (K73R + E166D). All four rate constants in the acyl-enzyme mechanism were determined for the E166D mutant by the methods described by Christensen, Martin & Waley [(1990) Biochem. J. 266, 853-861]. Both the rate constants for acylation and deacylation for the hydrolysis of benzylpenicillin were decreased about 2000-fold in this mutant. In the K73R mutant, and in the double mutant, the rate constants for acylation were decreased about 100-fold and 10000-fold respectively. All three mutants also had lowered values for the rate constants for the formation and dissociation of the non-covalent enzyme-substrate complex. The specificities of the mutants did not differ greatly from those of wild-type beta-lactamase, but the hydrolysis of cephalosporin C by the K73R mutant gave 'burst' kinetics.
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页码:613 / 619
页数:7
相关论文
共 59 条
[1]   COMPARISON OF THE ACTION OF PENICILLINASE ON BENZYLPENICILLIN AND CEPHALOSPORIN-N AND THE COMPETITIVE INHIBITION OF PENICILLINASE BY CEPHALOSPORIN-C [J].
ABRAHAM, EP ;
NEWTON, GGF .
BIOCHEMICAL JOURNAL, 1956, 63 (04) :628-634
[2]   CRYSTALLOGRAPHY AND SITE-DIRECTED MUTAGENESIS OF YEAST TRIOSEPHOSPHATE ISOMERASE - WHAT CAN WE LEARN ABOUT CATALYSIS FROM A SIMPLE ENZYME [J].
ALBER, TC ;
DAVENPORT, RC ;
GIAMMONA, DA ;
LOLIS, E ;
PETSKO, GA ;
RINGE, D .
COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY, 1987, 52 :603-613
[4]  
BAGULEY C, 1990, THESIS U OXFORD
[5]   FRACTIONAL DIFFUSION-LIMITED COMPONENT OF REACTIONS CATALYZED BY ACETYLCHOLINESTERASE [J].
BAZELYANSKY, M ;
ROBEY, E ;
KIRSCH, JF .
BIOCHEMISTRY, 1986, 25 (01) :125-130
[6]   SINGLE-TURNOVER AND STEADY-STATE KINETICS OF HYDROLYSIS OF CEPHALOSPORINS BY BETA-LACTAMASE-I FROM BACILLUS-CEREUS [J].
BICKNELL, R ;
WALEY, SG .
BIOCHEMICAL JOURNAL, 1985, 231 (01) :83-88
[7]   TRIOSEPHOSPHATE ISOMERASE CATALYSIS IS DIFFUSION CONTROLLED - APPENDIX - ANALYSIS OF TRIOSE PHOSPHATE EQUILIBRIA IN AQUEOUS-SOLUTION BY P-31 NMR [J].
BLACKLOW, SC ;
RAINES, RT ;
LIM, WA ;
ZAMORE, PD ;
KNOWLES, JR .
BIOCHEMISTRY, 1988, 27 (04) :1158-1167
[8]   KINETIC-PARAMETERS OF THE ACYL-ENZYME MECHANISM AND CONDITIONS FOR QUASI-EQUILIBRIUM AND FOR OPTIMAL CATALYTIC CHARACTERISTICS [J].
BROCKLEHURST, K ;
TOPHAM, CM .
BIOCHEMICAL JOURNAL, 1990, 270 (02) :561-563
[9]   EVOLUTIONARY OPTIMIZATION OF THE CATALYTIC EFFECTIVENESS OF AN ENZYME [J].
BURBAUM, JJ ;
RAINES, RT ;
ALBERY, WJ ;
KNOWLES, JR .
BIOCHEMISTRY, 1989, 28 (24) :9293-9305
[10]  
BURNETTE WN, 1981, ANAL BIOCHEM, V112, P195, DOI 10.1016/0003-2697(81)90281-5