pH Dependence of and Kinetic Solvent Isotope Effects on the Methanolysis and Hydrolysis of β-Lactams Catalyzed by Class C β-Lactamase

Michael I. Page, Bartolomé Vilanova, Nicola J. Layland

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

The methanolysis of benzylpenicillin is catalyzed by Enterobacter cloacae P99 class C β-lactamase and the pH dependence of kcat indicates that the catalytic groups involved of pKa ca. 5 and 10 are the same as those for hydrolysis. The kinetic solvent isotope effect (KSIE) is 1.42 for both the hydrolysis and methanolysis of benzylpenicillin. However, there is an inverse KSIE on of 0.83 ± 0.05 for the hydrolysis of benzylpenicillin and cephaloridine. There is also an abnormally high shift in the low pKa on going from H2O to D2O of 0.85 ± 0.15 from the pH dependence of both kcat and kcat/Km for both methanolysis and hydrolysis. The D2O shift on the high pKa of ca. 10 is the normal value of ca. 0.4. These results are consistent with a strongly hydrogen bonded system acting as the general base catalyst and low fractionation factors for the hydrogens involved. It is probable that this represents the tyrosine phenoxide ion hydrogen bonded to two lysine ammonium ions.

Original languageEnglish
Pages (from-to)12092-12095
Number of pages4
JournalJournal of the American Chemical Society
Volume117
Issue number49
DOIs
Publication statusPublished - Dec 1995

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Lactams
Isotopes
Penicillin G
Hydrolysis
Kinetics
Hydrogen
Cephaloridine
Enterobacter cloacae
Ions
Fractionation
Ammonium Compounds
Lysine
Tyrosine
Protons
Reference Values
Catalysts

Cite this

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title = "pH Dependence of and Kinetic Solvent Isotope Effects on the Methanolysis and Hydrolysis of β-Lactams Catalyzed by Class C β-Lactamase",
abstract = "The methanolysis of benzylpenicillin is catalyzed by Enterobacter cloacae P99 class C β-lactamase and the pH dependence of kcat indicates that the catalytic groups involved of pKa ca. 5 and 10 are the same as those for hydrolysis. The kinetic solvent isotope effect (KSIE) is 1.42 for both the hydrolysis and methanolysis of benzylpenicillin. However, there is an inverse KSIE on of 0.83 ± 0.05 for the hydrolysis of benzylpenicillin and cephaloridine. There is also an abnormally high shift in the low pKa on going from H2O to D2O of 0.85 ± 0.15 from the pH dependence of both kcat and kcat/Km for both methanolysis and hydrolysis. The D2O shift on the high pKa of ca. 10 is the normal value of ca. 0.4. These results are consistent with a strongly hydrogen bonded system acting as the general base catalyst and low fractionation factors for the hydrogens involved. It is probable that this represents the tyrosine phenoxide ion hydrogen bonded to two lysine ammonium ions.",
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pH Dependence of and Kinetic Solvent Isotope Effects on the Methanolysis and Hydrolysis of β-Lactams Catalyzed by Class C β-Lactamase. / Page, Michael I.; Vilanova, Bartolomé; Layland, Nicola J.

In: Journal of the American Chemical Society, Vol. 117, No. 49, 12.1995, p. 12092-12095.

Research output: Contribution to journalArticle

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N2 - The methanolysis of benzylpenicillin is catalyzed by Enterobacter cloacae P99 class C β-lactamase and the pH dependence of kcat indicates that the catalytic groups involved of pKa ca. 5 and 10 are the same as those for hydrolysis. The kinetic solvent isotope effect (KSIE) is 1.42 for both the hydrolysis and methanolysis of benzylpenicillin. However, there is an inverse KSIE on of 0.83 ± 0.05 for the hydrolysis of benzylpenicillin and cephaloridine. There is also an abnormally high shift in the low pKa on going from H2O to D2O of 0.85 ± 0.15 from the pH dependence of both kcat and kcat/Km for both methanolysis and hydrolysis. The D2O shift on the high pKa of ca. 10 is the normal value of ca. 0.4. These results are consistent with a strongly hydrogen bonded system acting as the general base catalyst and low fractionation factors for the hydrogens involved. It is probable that this represents the tyrosine phenoxide ion hydrogen bonded to two lysine ammonium ions.

AB - The methanolysis of benzylpenicillin is catalyzed by Enterobacter cloacae P99 class C β-lactamase and the pH dependence of kcat indicates that the catalytic groups involved of pKa ca. 5 and 10 are the same as those for hydrolysis. The kinetic solvent isotope effect (KSIE) is 1.42 for both the hydrolysis and methanolysis of benzylpenicillin. However, there is an inverse KSIE on of 0.83 ± 0.05 for the hydrolysis of benzylpenicillin and cephaloridine. There is also an abnormally high shift in the low pKa on going from H2O to D2O of 0.85 ± 0.15 from the pH dependence of both kcat and kcat/Km for both methanolysis and hydrolysis. The D2O shift on the high pKa of ca. 10 is the normal value of ca. 0.4. These results are consistent with a strongly hydrogen bonded system acting as the general base catalyst and low fractionation factors for the hydrogens involved. It is probable that this represents the tyrosine phenoxide ion hydrogen bonded to two lysine ammonium ions.

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