Structural Basis of Extended Spectrum TEM ␤-Lactamases CRYSTALLOGRAPHIC, KINETIC, AND MASS SPECTROMETRIC INVESTIGATIONS OF ENZYME MUTANTS* - CNRS - Centre national de la recherche scientifique Accéder directement au contenu
Article Dans Une Revue Journal of Biological Chemistry Année : 1996

Structural Basis of Extended Spectrum TEM ␤-Lactamases CRYSTALLOGRAPHIC, KINETIC, AND MASS SPECTROMETRIC INVESTIGATIONS OF ENZYME MUTANTS*

Isabelle Saves
  • Fonction : Auteur
Odile Burlet-Schiltz
Peter Swarén
  • Fonction : Auteur
Jean-Michel Masson
  • Fonction : Auteur
Myriam Delaire
  • Fonction : Auteur
Jean-Claude Promé
  • Fonction : Auteur
Jean-Pierre Samama
  • Fonction : Auteur

Résumé

The E166Y and the E166Y/R164S TEM-1 ␤-lactamase mutant enzymes display extended spectrum substrate specificities. Electrospray mass spectrometry demonstrates that, with penicillin G as substrate, the rate-limiting step in catalysis is the hydrolysis of the E166Y acyl-enzyme complex. Comparison of the 1.8-Å resolution x-ray structures of the wild-type and of the E166Y mutant enzymes shows that the binding of cephalo-sporin substrates is improved, in the mutant enzyme, by the enlargement of the substrate binding site. This enlargement is due to the rigid body displacement of 60 residues driven by the movement of the ⍀-loop. These structural observations strongly suggest that the link between the position of the ⍀-loop and that of helix H5, plays a central role in the structural events leading to extended spectrum TEM-related enzymes. The increased ⍀-loop flexibility caused by the R164S mutation, which is found in several natural mutant TEM enzymes, may lead to similar structural effects. Comparison of the kinetic data of the E166Y, E166Y/R164S, and R164S mutant enzymes supports this hypothesis.
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hal-03004742 , version 1 (20-11-2020)

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Laurent Maveyraud, Isabelle Saves, Odile Burlet-Schiltz, Peter Swarén, Jean-Michel Masson, et al.. Structural Basis of Extended Spectrum TEM ␤-Lactamases CRYSTALLOGRAPHIC, KINETIC, AND MASS SPECTROMETRIC INVESTIGATIONS OF ENZYME MUTANTS*. Journal of Biological Chemistry, 1996, 271 (18), pp.10482-10489. ⟨10.1074/jbc.271.18.10482⟩. ⟨hal-03004742⟩
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