Inactivation of Class A β-Lactamases by Clavulanic Acid

The Role of Arginine-244 in a Proposed Nonconcerted Sequence of Events

Uzma Imtiaz, Shahriar Mobashery, Eric Billings, Elias Kurian Manavathu, Elias K. Manavathu, Stephen A. Lerner, Stephen A. Lerner, Stephen A. Lerner

Research output: Contribution to journalArticle

118 Citations (Scopus)

Abstract

From the refined 2 Å crystal structure of the Bacillus licheniformis 749/C β-lactamase, energy-minimized models for active-site binding of the precatalytic (“Michaelis”) complex with the clinically utilized inactivator, clavulanic acid, for the acyl enzyme intermediate, and for the ultimate acylated acyclic species that leads to inactivation of class A β-lactamases by clavulanate have been generated. On the basis of these models, the details of the chemistry of inactivation of clavulanate are reassessed. A nonconcerted process for the inactivation chemistry of class A β-lactamases by clavulanate is proposed. These models reveal that the Arg-244 side chain and the Val-216 carbonyl anchor a structurally conserved water molecule, W673, which serves as the most likely source of a critical proton in a stepwise sequence of events. Disruption of this “electrostatic anchor” for W673 by mutational replacement of Arg-244 with Ser in the TEM β-lactamase would account for the resulting observed severe impairment of the efficiency of inactivation of the mutant enzyme by clavulanate. The kinetic impact of the Arg-244-Ser mutation on interaction with clavulanate is reflected by resistance to ampicillin plus clavulanate of a strain of E. coli bearing the mutant enzyme. Molecular dynamics computations on the acylated acyclic intermediate—the putative inactivating species—indicated that irreversible inactivation of the β-lactamase may not occur as a consequence of a transimination reaction, in contrast to previous suggestions. The most likely scenario for irreversible inactivation involves the capture of the β-hydroxyl of conserved Ser-130 by the iminium moiety of the acylated acyclic intermediate, followed by a deprotonation at C6 of clavulanate. The deprotonation is likely to be carried out by the conserved Glu-166 via the intervening crystallographic water W712. Deprotonation prior to nucleophile capture is proposed as the mechanism of generation of the so-called transiently inhibited enamine species. For the wild-type TEM-1 β-lactamase, both irreversible inactivation and the formation of the transiently inhibited species proceed with comparable rates. In addition, a new function for the Ser-130 in the formation of the acyl-enzyme intermediate with both clavulanate and typical β-lactamase substrates is proposed. It is suggested that the β-hydroxyl of Ser-130 stabilizes the transition state for the expulsion of the incipient amine from the high-energy tetrahedral species by hydrogen bonding to the oxazolidine amine in the course of Ser-70 acylation.

Original languageEnglish (US)
Pages (from-to)4435-4442
Number of pages8
JournalJournal of the American Chemical Society
Volume115
Issue number11
DOIs
StatePublished - May 1 1993

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Clavulanic Acid
Arginine
Deprotonation
Enzymes
Acids
Anchors
Amines
Bearings (structural)
Transmission electron microscopy
Acylation
Enzyme kinetics
Nucleophiles
Bacilli
Binding sites
Escherichia coli
Molecular dynamics
Water
Electrostatics
Hydroxyl Radical
Hydrogen bonds

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Imtiaz, U., Mobashery, S., Billings, E., Manavathu, E. K., Manavathu, E. K., Lerner, S. A., ... Lerner, S. A. (1993). Inactivation of Class A β-Lactamases by Clavulanic Acid: The Role of Arginine-244 in a Proposed Nonconcerted Sequence of Events. Journal of the American Chemical Society, 115(11), 4435-4442. https://doi.org/10.1021/ja00064a003

Inactivation of Class A β-Lactamases by Clavulanic Acid : The Role of Arginine-244 in a Proposed Nonconcerted Sequence of Events. / Imtiaz, Uzma; Mobashery, Shahriar; Billings, Eric; Manavathu, Elias Kurian; Manavathu, Elias K.; Lerner, Stephen A.; Lerner, Stephen A.; Lerner, Stephen A.

In: Journal of the American Chemical Society, Vol. 115, No. 11, 01.05.1993, p. 4435-4442.

Research output: Contribution to journalArticle

Imtiaz, U, Mobashery, S, Billings, E, Manavathu, EK, Manavathu, EK, Lerner, SA, Lerner, SA & Lerner, SA 1993, 'Inactivation of Class A β-Lactamases by Clavulanic Acid: The Role of Arginine-244 in a Proposed Nonconcerted Sequence of Events', Journal of the American Chemical Society, vol. 115, no. 11, pp. 4435-4442. https://doi.org/10.1021/ja00064a003
Imtiaz, Uzma ; Mobashery, Shahriar ; Billings, Eric ; Manavathu, Elias Kurian ; Manavathu, Elias K. ; Lerner, Stephen A. ; Lerner, Stephen A. ; Lerner, Stephen A. / Inactivation of Class A β-Lactamases by Clavulanic Acid : The Role of Arginine-244 in a Proposed Nonconcerted Sequence of Events. In: Journal of the American Chemical Society. 1993 ; Vol. 115, No. 11. pp. 4435-4442.
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