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PMID: 1980064 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Site-directed mutagenesis of beta-lactamase I. Single and double mutants of Glu-166 and Lys-73.

The Biochemical journal ·Vol. 272 ·No. 3 ·1990-12-15 ·Pages 613-9

Gibson RM, Christensen H, Waley SG

Abstract

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 10,000-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.

MeSH Terms
Bacillus cereus/enzymology,genetics Cephalosporins/metabolism Escherichia coli/genetics Glutamates Glutamic Acid Hydrolysis Kinetics Lysine Mutagenesis, Site-Directed Penicillin G/metabolism Penicillinase/genetics,metabolism Substrate Specificity
Chemicals
Cephalosporins Glutamates Glutamic Acid cephalosporin C Penicillinase Lysine Penicillin G
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Gibson R M
Sir William Dunn School of Pathology, University of Oxford, U.K.
Christensen H
Waley S G
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj2720613
Published
1990-12-15
Pages
613-9
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1149752
Subset
IM
External Links
PubMed source DOI: 10.1042/bj2720613 PMC Full Text View journal details
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