PK(A) CALCULATIONS FOR CLASS-A BETA-LACTAMASES - METHODOLOGICAL AND MECHANISTIC IMPLICATIONS

beta-lactamases are responsible for resistance to penicillins and rela ted beta-lactam compounds. Despite numerous studies, the identity of t he general base involved in the acylation step is still unclear. It ha s been proposed, on the basis of a previous pK(a) calculation and anal ysis of structural data, that the unprotonated Lys(73) in the active s ite could act as the general base. Using a continuum electrostatic mod el with an improved treatment of the multiple titration site problem, we calculated the pK(a) values of all titratable residues in the subst rate-free TEM-1 and Bacillus licheniformis class A beta-lactamases. Th e pK(a) of Lys(73) in both enzymes was computed to be above 10, in goo d agreement with recent experimental data on the TEM-1 beta-lactamase, but inconsistent with the proposal that Lys(73) acts as the general b ase. Even when the closest titratable residue, Glu(166), is mutated to a neutral residue, the predicted downward shift of the pK(a) of Lys(7 3) shows that it is unlikely to act as a proton abstractor in either e nzyme. These results support a mechanism in which the proton of the ac tive Ser(70) is transferred to the carboxylate group of Glu(166).