Electrostatic analysis of TEM1 -lactamase: effect of substrate binding, steep potential gradients and consequences of site-directed mutations

Background: Escherichia coli TEM1 is a penicillinase and belongs to class A-lactamases. Its naturally occurring mutants are responsible for bacterial resistance to 3-lactamin-based antibiotics. X-ray structure determina-tions show that all class A-lactamases are similar, but, despite the numerous kinetic investigations, the reaction mechanism of these enzymes is still debated. We address the questions of what the molecular contexts during the acylation and deacylation steps are and how they contribute to the efficiency of these penicillinases. Results: Electrostatic analysis of the 1.8 A resolution refined X-ray structure of the wild-type enzyme, and of its modelled Michaelis and acyl-enzyme complexes, showed that substrate binding induces an upward shift in the pKa of the unprotonated Lys73 by 6.4 pH units. The amine group of Lys73 can then abstract the Ser70 hydroxyl group proton and promote acylation. In the acyl-enzyme complex, the deacylating water is situated between the carboxylate group of Glu166, within the enzyme, and the ester-carbonyl carbon of the acyl-enzyme complex, in an elec-trostatic potential gradient amounting to 30 kTe-' A-'. Other residues, not directly involved in catalysis, also contribute to the formation of this gradient. The deacylation rate is related to the magnitude of the gradient. The kinetic behaviour of site-directed mutants that affect the protona-tion state of residue 73 cannot be explained on the basis of the wild-type enzyme mechanism. Conclusions: In the wild-type enzyme, the very high rates of acylation and deacylation of class A P-lactamases arise from an optimal chemical setup in which the acyla-tion reaction seems triggered by substrate binding that changes the general base property of Lys73. In site-directed mutants where Lys73 is protonated, acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuffle partner in this pathway.