Structure and Kinetics of the β-Lactamase Mutants S70A and K73H from Staphylococcus aureus PC1

• Published in: Biochemistry (Easton) 1996-09, Vol.35 (38), p.12251-12258
• Main Authors: Chen, Celia C, Smith, Tom J, Kapadia, Geeta, Wäsch, Susana, Zawadzke, Laura E, Coulson, Andrew, Herzberg, Osnat
• Format: Article
• Language: English
• Subjects: beta-Lactamases - chemistry; beta-Lactamases - genetics; beta-Lactamases - metabolism; Binding Sites; Cephalosporins - metabolism; Cloning, Molecular; Crystallography, X-Ray; DNA Primers; Escherichia coli - genetics; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Penicillin G - metabolism; Protein Conformation; Staphylococcus aureus - enzymology; Staphylococcus aureus - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi961153v

Two mutant β-lactamases from Staphylococcus aureus PC1 which probe key catalytic residues have been produced by site-directed mutagenesis. In the S70A enzyme, the nucleophilic group that attacks the β-lactam carbonyl carbon atom was eliminated. Consequently, the k cat values for hydrolysis of benzylpenicillin and nitrocefin have been reduced by 104−105 compared with the wild-type enzyme. The crystal structure of S70A β-lactamase has been determined at 2.1 Å 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 Å resolution. For both experiments, the difference electron density maps revealed buildup of density in the active site that presumably corresponds to β-lactam binding. However, neither electron density is sufficiently clear for defining the atomic details of the bound compounds. The K73H β-lactamase has been prepared to test the possible role of Lys73 in proton transfer. It exhibits no detectable activity toward benzylpenicillin, and 105-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 β-lactamase has been determined at 1.9 Å 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 Å resolution. The electron density map showed no indication of substrate binding.