Structure and kinetics of the beta-lactamase, mutants S70A and K73H from Staphylococcus aureus PC1

Two mutant beta-lactamases from Staphylococcus aureus PCl which probe key catalytic residues have been produced by site-directed mutagenesis. In the S70A enzyme, the nucleophilic group that attacks the beta-lactam carbonyl carbon atom was eliminated. Consequently, the k(cat) values for hydrolysis of benzylpenicillin and nitrocefin have been reduced by 10(4)-10(5) compared with the wild-type enzyme. The crystal structure of S70A beta-lactamase has been determined at 2.1 Angstrom resolution. With the exception of the mutation site, the structure is identical to that of the native enzyme. The residual activity is attributed either to mistranslation that leads to production of wild-type enzyme and/or to remaining features of the active site that stabilize the tetrahedral transition state. Soaking of the crystals with ampicillin or clavulanate, followed by flash-freezing, has been carried out and the structures examined at 2.0 Angstrom resolution. For both experiments, the difference electron density maps revealed buildup of density in the active site that presumably corresponds to beta-lactam binding, However, neither electron density is sufficiently clear for defining the atomic details of the bound compounds The K73H beta-lactamase has been prepared to test the possible role of Lys73 in proton transfer. It exhibits no detectable activity toward benzylpenicillin, and 10(5)-fold reduction of k(cat) for nitrocefin hydrolysis compared with the wild-type enzyme. No significant recovery of activity has been measured when the pH was varied between 5.0 and 8.0, The crystal structure of K73H beta-lactamase has been determined at 1.9 Angstrom resolution. While the overall structure is similar to that of the native enzyme, the electrostatic interactions between His73 and neighboring residues indicate that the imidazole ring is positively charged. In addition, the hydroxyl group of Ser70 adopts a position that is incompatible with nucleophilic attack on substrates. A crystal soaked with ampicillin was flash-frozen, and diffraction data were collected at 2.1 Angstrom resolution. The electron density map showed no indication of substrate binding.