Loss of enzyme activity during turnover of the Bacillus cereus beta-lactamase catalysed hydrolysis of beta-lactams due to loss of zinc ion.
Metallo-beta-lactamases are zinc-ion-dependent and are known to exist either as mononuclear or as dinuclear enzymes. The kinetics and mechanism of hydrolysis of the native zinc Bacillus cereus metallo-beta-lactamase (BcII) have been investigated under pre-steady-state conditions at different pHs and zinc-ion concentrations. Biphasic kinetics are observed for the hydrolysis of cefuroxime and benzylpenicillin with submicromolar concentrations of enzyme and zinc. The initial burst of product formation far exceeds the concentration of enzyme and the subsequent slower rate of hydrolysis is attributed to a branched kinetic pathway. The pH and metal-ion dependence of the microscopic rate constants of this branching were determined, from which it is concluded that two enzyme species with different metal-to-enzyme stoichiometries are formed during catalytic turnover. The dizinc enzyme is responsible for the fast route but during the catalytic cycle it slowly loses the less tightly bound zinc ion via the branching route to give an inactive monozinc enzyme; the latter is only catalytic following the uptake of a second zinc ion. The rate constant for product formation from the dinuclear enzyme and the branching rate constant show a sigmoidal dependence on pH indicative of important ionizing groups with pK(a)s of 9.0+/-0.1 and 8.2+/-0.1, respectively. The rate constant for the regeneration of enzyme activity depends on zinc-ion concentration. This unusual behaviour is attributed to an intrinsic property of metallo hydrolytic enzymes that depend on a metal bound water both as a ligand for the second metal ion and as the nucleophile which is consumed during hydrolysis of the substrate and so has to be replaced to maintain the catalytic cycle.
Badarau
Adriana
A
Department of Chemical and Biological Sciences, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, UK.
Page
Michael I
MI
D001409:Bacillus cereus / Q000201:enzymology*; D002384:Catalysis; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007477:Ions / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D012680:Sensitivity and Specificity; D013997:Time Factors; D015032:Zinc / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry*; D047090:beta-Lactams / Q000737:chemistry*
D007477:Ions; D047090:beta-Lactams; D001618:beta-Lactamases; D015032:Zinc