title: pKa calculations for class A beta-lactamases: methodological and mechanistic implications.
abstract: Beta-lactamases are responsible for resistance to penicillins and related beta-lactam compounds. Despite numerous studies, the identity of the general base involved in the acylation step is still unclear. It has been proposed, on the basis of a previous pKa calculation and analysis of structural data, that the unprotonated Lys73 in the active site could act as the general base. Using a continuum electrostatic model with an improved treatment of the multiple titration site problem, we calculated the pKa values of all titratable residues in the substrate-free TEM-1 and Bacillus licheniformis class A beta-lactamases. The pKa of Lys73 in both enzymes was computed to be above 10, in good agreement with recent experimental data on the TEM-1 beta-lactamase, but inconsistent with the proposal that Lys73 acts as the general base. Even when the closest titratable residue, Glu166, is mutated to a neutral residue, the predicted downward shift of the pKa of Lys73 shows that it is unlikely to act as a proton abstractor in either enzyme. These results support a mechanism in which the proton of the active Ser70 is transferred to the carboxylate group of Glu166.
authors.affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: X
authors.initials: X
authors.lastname: Raquet
authors.forename: V
authors.initials: V
authors.lastname: Lounnas
authors.forename: J
authors.initials: J
authors.lastname: Lamotte-Brasseur
authors.forename: J M
authors.initials: JM
authors.lastname: Frère
authors.forename: R C
authors.initials: RC
authors.lastname: Wade
mesh_terms: D000215:Acylation; D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011522:Protons; D012984:Software; D055672:Static Electricity; D017186:Titrimetry; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011522:Protons; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1