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pKa Calculations for class A β-lactamases: Influence of substrate binding

Published online by Cambridge University Press:  01 February 1999

JOSETTE LAMOTTE-BRASSEUR
Affiliation:
Centre d'Ingénierie des Protéines, University of Liège, 4000, Sart-Tilman, Belgium European Molecular Biology Laboratory, D-69117, Heidelberg, Germany
VALÈRE LOUNNAS
Affiliation:
European Molecular Biology Laboratory, D-69117, Heidelberg, Germany
XAVIER RAQUET
Affiliation:
Max-Delbrück Laboratorium, Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-weg 10, D-50829, Cologne, Germany
REBECCA C. WADE
Affiliation:
European Molecular Biology Laboratory, D-69117, Heidelberg, Germany
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Abstract

β-Lactamases are responsible for bacterial resistance to β-lactams and are thus of major clinical importance. However, the identity of the general base involved in their mechanism of action is still unclear. Two candidate residues, Glu166 and Lys73, have been proposed to fulfill this role. Previous studies support the proposal that Glu166 acts during the deacylation, but there is no consensus on the possible role of this residue in the acylation step. Recent experimental data and theoretical considerations indicate that Lys73 is protonated in the free β-lactamases, showing that this residue is unlikely to act as a proton abstractor. On the other hand, it has been proposed that the pKa of Lys73 would be dramatically reduced upon substrate binding and would thus be able to act as a base. To check this hypothesis, we performed continuum electrostatic calculations for five wild-type and three β-lactamase mutants to estimate the pKa of Lys73 in the presence of substrates, both in the Henri–Michaelis complex and in the tetrahedral intermediate. In all cases, the pKa of Lys73 was computed to be above 10, showing that it is unlikely to act as a proton abstractor, even when a β-lactam substrate is bound in the enzyme active site. The pKa of Lys234 is also raised in the tetrahedral intermediate, thus confirming a probable role of this residue in the stabilization of the tetrahedral intermediate. The influence of the β-lactam carboxylate on the pKa values of the active-site lysines is also discussed.

Type
Research Article
Copyright
© 1999 The Protein Society

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