pK(a) calculations for class A beta-lactamases: Influence of substrate binding

beta-Lactamases are responsible for bacterial resistance to beta-lactams an d are thus of major clinical importance. However, the identity of the gener al base involved in their mechanism of action is still unclear. Two candida te residues, Glu166 and Lys73, have been proposed to fulfill this role. Pre vious studies support the proposal that Glu166 acts during the deacylation. but there is no consensus on the possible role of this residue in the acyl ation step. Recent experimental data and theoretical considerations indicat e that Lys73 is protonated in the free beta-lactamases, showing that this r esidue is unlikely to act as a proton abstractor On the other hand, it has been proposed that the pK(a) of Lys73 would be dramatically reduced upon su bstrate binding and would thus be able to act as a base. To check this hypo thesis, we performed continuum electrostatic calculations for five wild-typ e and three beta-lactamase mutants to estimate the pK(a) of Lys73 in the pr esence of substrates, both in the Henri-Michaelis complex and in the tetrah edral intermediate. In all cases, the pK(a) of Lys73 was computed to be abo ve 10, showing that it is unlikely to act as a proton abstractor, even when a beta-lactam substrate is bound in the enzyme active site. The pK(a) of L ys234 is also raised in the tetrahedral intermediate, thus confirming a pro bable role of this residue in the stabilization of the tetrahedral intermed iate. The influence of the beta-lactam carboxylate on the pK(a) values of t he active-site lysines is also discussed.