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.
Research Article| December 15 1990
Site-directed mutagenesis of β-lactamase I. Single and double mutants of Glu-166 and Lys-73
R M Gibson;
Biochem J (1990) 272 (3): 613–619.
- Views Icon Views
- PDF LinkPDF
- Share Icon Share
- Cite Icon Cite
R M Gibson, H Christensen, S G Waley; Site-directed mutagenesis of β-lactamase I. Single and double mutants of Glu-166 and Lys-73. Biochem J 15 December 1990; 272 (3): 613–619. doi: https://doi.org/10.1042/bj2720613
Download citation file: