Molecular Dynamics Simulation of a Class A .beta.-Lactamase: Structural and Mechanistic Implications

Vijayakumar, S.; Ravishanker, G.; Pratt, Robertson; Beveridge, D. L.

doi:10.1021/ja00111a008

Cited by 41 publications

(61 citation statements)

References 3 publications

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“…Deacylation would then occur according to a nearly symmetrical pathway, with the intervention of a second, exogenous, water molecule (6). An alternative, recently proposed, hypothesis is that the flexibility of the "omega-loop," on which Glu-166 is situated, might be sufficient to bring its side chain near the Ser-70 hydroxyl (32).…”

Section: Discussionmentioning

confidence: 99%

The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme.

Damblon

Raquet

Lian

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

116

110

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ABSTRACT,B-Lactamases are widespread in the bacterial world, where they are responsible for resistance to penicillins, cephalosporins, and related compounds, currently the most widely used antibacterial agents. Detailed structural and mechanistic understanding of these enzymes can be expected to guide the design of new antibacterial compounds resistant to their action. A number of high-resolution structures are available for class A 8-lactamases, whose catalytic mechanism involves the acylation of a serine residue at the active site. The identity of the general base which participates in the activation of this serine residue during catalysis has been the subject of controversy, both a lysine residue and a glutamic acid residue having been proposed as candidates for this role. We have used the pH dependence of chemical modification of e-amino groups by 2,4,6,-trinitrobenzenesulfonate and the pH dependence of the e-methylene 'H and '3C chemical shifts (in enzyme selectively labeled with [E-'3C]lysine) to estimate the pKa of the relevant lysine residue, lysine-73, of TEM-1 ,B-lactamase. Both methods show that the pKa of this residue is > 10,

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Section: Discussionmentioning

confidence: 99%

The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme.

Damblon

Raquet

Lian

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

116

110

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“…The fact that acylation occurs with N170L ␤-lactamase (although probably much more slowly than with wild type) is not consistent with a mechanism involving E166 acting as a general base to activate Ser-70 via the hydrolytic water molecule (26). Since acylation is observed with mutations at E166 (5,10,27,28), it is clear that if this residue is normally involved in acylation there must be alternate pathways.…”

Section: Proposed Catalytic Mechanism Leading To Formation Of D-55-dmentioning

confidence: 96%

A point mutation leads to altered product specificity in β-lactamase catalysis

Lewis

Winterberg

Fink

1997

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACT␤-Lactamases are the primary cause of ␤-lactam antibiotic resistance in many pathogenic organisms. The ␤-lactamase catalytic mechanism has been shown to involve a covalent acyl-enzyme. Examination of the structure of the class A ␤-lactamase from Bacillus licheniformis suggested that replacement of Asn-170 by leucine would disrupt the deacylation reaction by displacing the hydrolytic water molecule. When N170L ␤-lactamase was reacted with penicillins, a novel product was formed. We postulate that with leucine at position 170 the acyl-enzyme undergoes deacylation by an intramolecular rearrangement (rather than hydrolysis) to form a thiazolidine-oxazolinone as the initial product. The oxazolinone subsequently undergoes rapid breakdown leading to the formation of N-phenylacetylglycine and N-formylpenicillamine. This appears to be the first reported case where a point mutation leads to a change in enzyme mechanism resulting in a substantially altered product, effectively changing the product specificity of ␤-lactamase into that of D-Ala-D-Ala-carboxypeptidase interacting with benzylpenicillin.

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“…This change allows more flexibility in the loop, which in turn opens more space for bulky ␤-lactam substituents. Of equal importance is the likelihood that the carboxylic acid side chain of Glu-166 will fluctuate about its native position, where it must function as a general acid-base (21,91). The R164S TEM-1 mutant prepared by site-directed mutagenesis showed improved k cat /K m for cefotaxime, ceftazidime, and aztreonam (80).…”

Section: Residue 104 (Glu and Asp) (I) Environmentmentioning

confidence: 99%

Extended-spectrum and inhibitor-resistant TEM-type beta-lactamases: mutations, specificity, and three-dimensional structure

Knox

1995

Antimicrob Agents Chemother

294

339

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Molecular Dynamics Simulation of a Class A .beta.-Lactamase: Structural and Mechanistic Implications

Cited by 41 publications

References 3 publications

The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme.

The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme.

A point mutation leads to altered product specificity in β-lactamase catalysis

Extended-spectrum and inhibitor-resistant TEM-type beta-lactamases: mutations, specificity, and three-dimensional structure

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