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-lac tam carbonyl carbon atom was eliminated. Consequently, the k(cat) valu es for hydrolysis of benzylpenicillin and nitrocefin have been reduced by 10(4)-10(5) compared with the wild-type enzyme. The crystal struct ure of S70A beta-lactamase has been determined at 2.1 Angstrom resolut ion. With the exception of the mutation site, the structure is identic al to that of the native enzyme. The residual activity is attributed e ither to mistranslation that leads to production of wild-type enzyme a nd/or to remaining features of the active site that stabilize the tetr ahedral transition state. Soaking of the crystals with ampicillin or c lavulanate, followed by flash-freezing, has been carried out and the s tructures examined at 2.0 Angstrom resolution. For both experiments, t he difference electron density maps revealed buildup of density in the active site that presumably corresponds to beta-lactam binding, Howev er, neither electron density is sufficiently clear for defining the at omic details of the bound compounds The K73H beta-lactamase has been p repared to test the possible role of Lys73 in proton transfer. It exhi bits no detectable activity toward benzylpenicillin, and 10(5)-fold re duction of k(cat) for nitrocefin hydrolysis compared with the wild-typ e enzyme. No significant recovery of activity has been measured when t he pH was varied between 5.0 and 8.0, The crystal structure of K73H be ta-lactamase has been determined at 1.9 Angstrom resolution. While the overall structure is similar to that of the native enzyme, the electr ostatic interactions between His73 and neighboring residues indicate t hat the imidazole ring is positively charged. In addition, the hydroxy l group of Ser70 adopts a position that is incompatible with nucleophi lic attack on substrates. A crystal soaked with ampicillin was flash-f rozen, and diffraction data were collected at 2.1 Angstrom resolution. The electron density map showed no indication of substrate binding.