ELECTROSTATIC ANALYSIS OF TEM1 BETA-LACTAMASE - EFFECT OF SUBSTRATE-BINDING, STEEP POTENTIAL GRADIENTS AND CONSEQUENCES OF SITE-DIRECTED MUTATIONS

Background: Escherichia coli TEM1 is a penicillinase and belongs to cl ass A beta-lactamases. Its naturally occurring mutants are responsible for bacterial resistance to beta-lactamin-based antibiotics. X-ray st ructure determinations show that all class A beta-lactamases are simil ar, but, despite the numerous kinetic investigations, the reaction mec hanism 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 Angstrom resolution refin ed X-ray structure of the wild-type enzyme, and of its modelled Michae lis and acyl-enzyme complexes, showed that substrate binding induces a n upward shift in the pK(a) of the unprotonated Lys73 by 6.4 pH units. The amine group of Lys73 can then abstract the Ser70 hydroxyl group p roton and promote acylation. In the acyl-enzyme complex, the deacylati ng water is situated between the carboxylate group of Glu166, within t he enzyme, and the ester-carbonyl carbon of the acyl-enzyme complex, i n an electrostatic potential gradient amounting to 30 kTe(-1) Angstrom (-1). Other residues, not directly involved in catalysis, also contrib ute to the formation df this gradient. The deacylation rate is related to the magnitude of the gradient. The kinetic behaviour of site-direc ted mutants that affect the protonation 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 deacyla tion of class A beta-lactamases arise from an optimal chemical setup i n which the acylation reaction seems triggered by substrate binding th at changes the general base property of Lys73. In site-directed mutant s where Lys73 is protonated, acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuff le partner in this pathway.