Kinetic parameters of the acyl-enzyme mechanism

Waley, S. G.

doi:10.1042/bj2700563a

Cited by 1 publication

(1 citation statement)

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“…'Efficiency ' of mutants The results in Table 1 reveal the unexpected finding that all three mutants have comparable k-1 and k+2 values. This feature, which was observed for wild-type ,J-lactamase I and for two other ,3-lactamases, was regarded as a sign of a 'fully efficient' enzyme (Christensen et al, 1990; but see Brocklehurst & Topham, 1990;Waley, 1990). The mutants that we are discussing here are clearly not fully efficient: this is obvious both from the diminished values of the kinetic parameters and from their diminished power to protect E. coli from ampicillin (R. M. Gibson & S. G. Waley, unpublished work).…”

Section: Effect Of Mutations On Enzyme-substrate Combinationmentioning

confidence: 99%

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

Gibson

Christensen

Waley

1990

Biochemical Journal

133

153

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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.

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Section: Effect Of Mutations On Enzyme-substrate Combinationmentioning

confidence: 99%