Loss of enzyme activity during turnover of the Bacillus cereus β-lactamase catalysed hydrolysis of β-lactams due to loss of zinc ion

Adriana Badarau, Michael I. Page

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Metallo-β-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-β-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 as 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.

Original languageEnglish
Pages (from-to)919-928
Number of pages10
JournalJournal of Biological Inorganic Chemistry
Volume13
Issue number6
Early online date1 May 2008
DOIs
Publication statusPublished - Aug 2008

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Bacillus cereus
Lactams
Enzyme activity
Zinc
Hydrolysis
Ions
Enzymes
Rate constants
Metals
Kinetics
Metal ions
1,4-alpha-Glucan Branching Enzyme
Cefuroxime
Nucleophiles
Penicillin G
Stoichiometry
Regeneration
Ligands
Water

Cite this

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title = "Loss of enzyme activity during turnover of the Bacillus cereus β-lactamase catalysed hydrolysis of β-lactams due to loss of zinc ion",
abstract = "Metallo-β-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-β-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 as 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.",
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Loss of enzyme activity during turnover of the Bacillus cereus β-lactamase catalysed hydrolysis of β-lactams due to loss of zinc ion. / Badarau, Adriana; Page, Michael I.

In: Journal of Biological Inorganic Chemistry, Vol. 13, No. 6, 08.2008, p. 919-928.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Loss of enzyme activity during turnover of the Bacillus cereus β-lactamase catalysed hydrolysis of β-lactams due to loss of zinc ion

AU - Badarau, Adriana

AU - Page, Michael I.

PY - 2008/8

Y1 - 2008/8

N2 - Metallo-β-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-β-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 as 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.

AB - Metallo-β-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-β-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 as 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.

KW - β-Lactams

KW - Antibiotics

KW - Biphasic kinetics

KW - Metalloenzymes

KW - Mutation

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U2 - 10.1007/s00775-008-0379-2

DO - 10.1007/s00775-008-0379-2

M3 - Article

VL - 13

SP - 919

EP - 928

JO - Journal of Biological Inorganic Chemistry

JF - Journal of Biological Inorganic Chemistry

SN - 0949-8257

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ER -