title: Site-directed mutagenesis of beta-lactamase I. Single and double mutants of Glu-166 and Lys-73.
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.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, U.K.
authors.forename: R M
authors.initials: RM
authors.lastname: Gibson
authors.forename: H
authors.initials: H
authors.lastname: Christensen
authors.forename: S G
authors.initials: SG
authors.lastname: Waley
mesh_terms: D001409:Bacillus cereus / Q000201:enzymology / Q000235:genetics; D002511:Cephalosporins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005971:Glutamates; D018698:Glutamic Acid; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D010400:Penicillin G / Q000378:metabolism; D010405:Penicillinase / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D002511:Cephalosporins; D005971:Glutamates; D018698:Glutamic Acid; C025163:cephalosporin C; D010405:Penicillinase; D008239:Lysine; D010400:Penicillin G