The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase

Sakina Bounaga, Andrew P. Laws, Moreno Galleni, Michael I. Page

Research output: Contribution to journalArticle

152 Citations (Scopus)

Abstract

The plot of k(cat)/K(m) against pH for the Bacillus cereus 569/H β-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pK(a) 5.6 ± 0.2 and one of pK(a) 9.5 ± 0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis. These are assigned to zinc(II)-bound water and Asp-90, both with a pK(a) of 5.6 ± 0.2. A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a K(i) of 70 μM. The pH-dependence of K(i) shows similar pH inflections to those observed in the catalysed hydrolysis of substrates. The pH-independence of K(i) between pH 6 and 9 indicates that the pK(a) of zinc(II)-bound water must be 5.6 and not the higher pK(a) of 9.5. The kinetic solvent isotope effect on k(cat)/K(m) is 1.3 ± 0.5 and that on k(cat) is 1.5. There is no effect on reactivity by either added zinc(II) or methanol. The possible mechanisms of action for the class B β-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.

Original languageEnglish
Pages (from-to)703-711
Number of pages9
JournalBiochemical Journal
Volume331
Issue number3
DOIs
Publication statusPublished - 1 May 1998

Fingerprint

Bacillus cereus
Catalysis
Zinc
Hydrolysis
pH
Lewis Acids
Nucleophiles
Penicillin G
Water
Cephalosporins
Sulfhydryl Compounds
Isotopes
Anions
Methanol
Catalysts
Kinetics
Acids
Substrates
Enzymes

Cite this

Bounaga, Sakina ; Laws, Andrew P. ; Galleni, Moreno ; Page, Michael I. / The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase. In: Biochemical Journal. 1998 ; Vol. 331, No. 3. pp. 703-711.
@article{35187c25328c41cf8330e54941df50d7,
title = "The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase",
abstract = "The plot of k(cat)/K(m) against pH for the Bacillus cereus 569/H β-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pK(a) 5.6 ± 0.2 and one of pK(a) 9.5 ± 0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis. These are assigned to zinc(II)-bound water and Asp-90, both with a pK(a) of 5.6 ± 0.2. A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a K(i) of 70 μM. The pH-dependence of K(i) shows similar pH inflections to those observed in the catalysed hydrolysis of substrates. The pH-independence of K(i) between pH 6 and 9 indicates that the pK(a) of zinc(II)-bound water must be 5.6 and not the higher pK(a) of 9.5. The kinetic solvent isotope effect on k(cat)/K(m) is 1.3 ± 0.5 and that on k(cat) is 1.5. There is no effect on reactivity by either added zinc(II) or methanol. The possible mechanisms of action for the class B β-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.",
author = "Sakina Bounaga and Laws, {Andrew P.} and Moreno Galleni and Page, {Michael I.}",
year = "1998",
month = "5",
day = "1",
doi = "10.1042/bj3310703",
language = "English",
volume = "331",
pages = "703--711",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "3",

}

The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase. / Bounaga, Sakina; Laws, Andrew P.; Galleni, Moreno; Page, Michael I.

In: Biochemical Journal, Vol. 331, No. 3, 01.05.1998, p. 703-711.

Research output: Contribution to journalArticle

TY - JOUR

T1 - The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase

AU - Bounaga, Sakina

AU - Laws, Andrew P.

AU - Galleni, Moreno

AU - Page, Michael I.

PY - 1998/5/1

Y1 - 1998/5/1

N2 - The plot of k(cat)/K(m) against pH for the Bacillus cereus 569/H β-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pK(a) 5.6 ± 0.2 and one of pK(a) 9.5 ± 0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis. These are assigned to zinc(II)-bound water and Asp-90, both with a pK(a) of 5.6 ± 0.2. A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a K(i) of 70 μM. The pH-dependence of K(i) shows similar pH inflections to those observed in the catalysed hydrolysis of substrates. The pH-independence of K(i) between pH 6 and 9 indicates that the pK(a) of zinc(II)-bound water must be 5.6 and not the higher pK(a) of 9.5. The kinetic solvent isotope effect on k(cat)/K(m) is 1.3 ± 0.5 and that on k(cat) is 1.5. There is no effect on reactivity by either added zinc(II) or methanol. The possible mechanisms of action for the class B β-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.

AB - The plot of k(cat)/K(m) against pH for the Bacillus cereus 569/H β-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pK(a) 5.6 ± 0.2 and one of pK(a) 9.5 ± 0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis. These are assigned to zinc(II)-bound water and Asp-90, both with a pK(a) of 5.6 ± 0.2. A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a K(i) of 70 μM. The pH-dependence of K(i) shows similar pH inflections to those observed in the catalysed hydrolysis of substrates. The pH-independence of K(i) between pH 6 and 9 indicates that the pK(a) of zinc(II)-bound water must be 5.6 and not the higher pK(a) of 9.5. The kinetic solvent isotope effect on k(cat)/K(m) is 1.3 ± 0.5 and that on k(cat) is 1.5. There is no effect on reactivity by either added zinc(II) or methanol. The possible mechanisms of action for the class B β-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.

UR - http://www.scopus.com/inward/record.url?scp=0032080039&partnerID=8YFLogxK

U2 - 10.1042/bj3310703

DO - 10.1042/bj3310703

M3 - Article

VL - 331

SP - 703

EP - 711

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 3

ER -