Enzyme deactivation due to metal-ion dissociation during turnover of the cobalt-β-lactamase catalyzed hydrolysis of β-lactams

Adriana Badarau, Michael I. Page

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Metallo-β-lactamases are native zinc enzymes that catalyze the hydrolysis of β-lactam antibiotics but are also able to function with cobalt (II) and require one or two metal ions for catalytic activity. The kinetics of the hydrolysis of benzylpenicillin catalyzed by cobalt substituted β-lactamase from Bacillus cereus (BcII) are biphasic. The dependence of enzyme activity on pH and metal-ion concentration indicates that only the di-cobalt enzyme is catalytically active. A mono-cobalt enzyme species is formed during the catalytic cycle, which is virtually inactive and requires the association of another cobalt ion for turnover. Two intermediates with different metal to enzyme stoichiometries are formed on a branched reaction pathway. The di-cobalt enzyme intermediate is responsible for the direct catalytic route, which is pH-independent between 5.5 and 9.5 but is also able to slowly lose one bound cobalt ion via the branching route to give the mono-cobalt inactive enzyme intermediate. This inactivation pathway of metal-ion dissociation occurs by both an acid catalyzed and a pH-independent reaction, which is dependent on the presence of an enzyme residue of pKa = 8.9 ± 0.1 in its protonated form and shows a large kinetic solvent isotope effect (H 2O/D2O) of 5.2 ± 0.5, indicative of a rate-limiting proton transfer. The pseudo first-order rate constant to regenerate the di-cobalt β-lactamase from the mono-cobalt enzyme intermediate has a first-order dependence on cobalt-ion concentration in the pH range 5.5-9.5. The second-order rate constant for metal-ion association is dependent on two groups of pKa 6.32 ± 0.1 and 7.47 ± 0.1 being in their deprotonated basic forms and one group of pKa 9.48 ± 0.1 being in its protonated form.

LanguageEnglish
Pages11012-11020
Number of pages9
JournalBiochemistry
Volume45
Issue number36
Early online date18 Aug 2006
DOIs
Publication statusPublished - 12 Sep 2006

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beta-Lactams
beta-Lactamases
Cobalt
Metal ions
Hydrolysis
Metals
Ions
Enzymes
Rate constants
Association reactions
Bacillus cereus
Kinetics
Proton transfer
Penicillin G
Enzyme activity
Isotopes
Stoichiometry
Protons
Zinc
Catalyst activity

Cite this

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title = "Enzyme deactivation due to metal-ion dissociation during turnover of the cobalt-β-lactamase catalyzed hydrolysis of β-lactams",
abstract = "Metallo-β-lactamases are native zinc enzymes that catalyze the hydrolysis of β-lactam antibiotics but are also able to function with cobalt (II) and require one or two metal ions for catalytic activity. The kinetics of the hydrolysis of benzylpenicillin catalyzed by cobalt substituted β-lactamase from Bacillus cereus (BcII) are biphasic. The dependence of enzyme activity on pH and metal-ion concentration indicates that only the di-cobalt enzyme is catalytically active. A mono-cobalt enzyme species is formed during the catalytic cycle, which is virtually inactive and requires the association of another cobalt ion for turnover. Two intermediates with different metal to enzyme stoichiometries are formed on a branched reaction pathway. The di-cobalt enzyme intermediate is responsible for the direct catalytic route, which is pH-independent between 5.5 and 9.5 but is also able to slowly lose one bound cobalt ion via the branching route to give the mono-cobalt inactive enzyme intermediate. This inactivation pathway of metal-ion dissociation occurs by both an acid catalyzed and a pH-independent reaction, which is dependent on the presence of an enzyme residue of pKa = 8.9 ± 0.1 in its protonated form and shows a large kinetic solvent isotope effect (H 2O/D2O) of 5.2 ± 0.5, indicative of a rate-limiting proton transfer. The pseudo first-order rate constant to regenerate the di-cobalt β-lactamase from the mono-cobalt enzyme intermediate has a first-order dependence on cobalt-ion concentration in the pH range 5.5-9.5. The second-order rate constant for metal-ion association is dependent on two groups of pKa 6.32 ± 0.1 and 7.47 ± 0.1 being in their deprotonated basic forms and one group of pKa 9.48 ± 0.1 being in its protonated form.",
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Enzyme deactivation due to metal-ion dissociation during turnover of the cobalt-β-lactamase catalyzed hydrolysis of β-lactams. / Badarau, Adriana; Page, Michael I.

In: Biochemistry, Vol. 45, No. 36, 12.09.2006, p. 11012-11020.

Research output: Contribution to journalArticle

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AU - Page, Michael I.

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AB - Metallo-β-lactamases are native zinc enzymes that catalyze the hydrolysis of β-lactam antibiotics but are also able to function with cobalt (II) and require one or two metal ions for catalytic activity. The kinetics of the hydrolysis of benzylpenicillin catalyzed by cobalt substituted β-lactamase from Bacillus cereus (BcII) are biphasic. The dependence of enzyme activity on pH and metal-ion concentration indicates that only the di-cobalt enzyme is catalytically active. A mono-cobalt enzyme species is formed during the catalytic cycle, which is virtually inactive and requires the association of another cobalt ion for turnover. Two intermediates with different metal to enzyme stoichiometries are formed on a branched reaction pathway. The di-cobalt enzyme intermediate is responsible for the direct catalytic route, which is pH-independent between 5.5 and 9.5 but is also able to slowly lose one bound cobalt ion via the branching route to give the mono-cobalt inactive enzyme intermediate. This inactivation pathway of metal-ion dissociation occurs by both an acid catalyzed and a pH-independent reaction, which is dependent on the presence of an enzyme residue of pKa = 8.9 ± 0.1 in its protonated form and shows a large kinetic solvent isotope effect (H 2O/D2O) of 5.2 ± 0.5, indicative of a rate-limiting proton transfer. The pseudo first-order rate constant to regenerate the di-cobalt β-lactamase from the mono-cobalt enzyme intermediate has a first-order dependence on cobalt-ion concentration in the pH range 5.5-9.5. The second-order rate constant for metal-ion association is dependent on two groups of pKa 6.32 ± 0.1 and 7.47 ± 0.1 being in their deprotonated basic forms and one group of pKa 9.48 ± 0.1 being in its protonated form.

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