Structure and Kinetics of the -Lactamase Mutants S70A and K73H from Staphylococcus aureus PC1
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- 作者：Celia C. Chen ; Tom J. Smith ; Geeta Kapadia ; Susana Wä ; sch ; Laura E. Zawadzke ; Andrew Coulson ; and Osnat Herzberg
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：September 24, 1996
- 年：1996
- 卷：35
- 期：38
- 页码：12251 - 12258
- 全文大小：474K
- 年卷期：v.35,no.38(September 24, 1996)
- ISSN：1520-4995
文摘
Two mutant 
-lactamases from Staphylococcusaureus PC1 which probe key catalytic residueshave been produced by site-directed mutagenesis. In the S70Aenzyme, the nucleophilic group that attacksthe -lactam carbonyl carbon atom was eliminated. Consequently,the kcat values for hydrolysis ofbenzylpenicillin and nitrocefin have been reduced by104-105 compared with the wild-typeenzyme.The crystal structure of S70A -lactamase has been determined at2.1 Å resolution. With the exceptionof the mutation site, the structure is identical to that of the nativeenzyme. The residual activity is attributedeither to mistranslation that leads to production of wild-type enzymeand/or to remaining features of theactive site that stabilize the tetrahedral transition state.Soaking of the crystals with ampicillin orclavulanate,followed by flash-freezing, has been carried out and the structuresexamined at 2.0 Å resolution. Forboth experiments, the difference electron density maps revealed buildupof density in the active site thatpresumably corresponds to -lactam binding. However, neitherelectron density is sufficiently clear fordefining the atomic details of the bound compounds. The K73H-lactamase has been prepared to testthe possible role of Lys73 in proton transfer. It exhibits nodetectable activity toward benzylpenicillin,and 105-fold reduction of kcat fornitrocefin hydrolysis compared with the wild-type enzyme. Nosignificantrecovery of activity has been measured when the pH was varied between5.0 and 8.0. The crystal structureof K73H -lactamase has been determined at 1.9 Å resolution.While the overall structure is similar tothat of the native enzyme, the electrostatic interactions between His73and neighboring residues indicatethat the imidazole ring is positively charged. In addition, thehydroxyl group of Ser70 adopts a positionthat is incompatible with nucleophilic attack on substrates. Acrystal soaked with ampicillin was flash-frozen, and diffraction data were collected at 2.1 Å resolution.The electron density map showed noindication of substrate binding.
-lactamases from Staphylococcusaureus PC1 which probe key catalytic residueshave been produced by site-directed mutagenesis. In the S70Aenzyme, the nucleophilic group that attacksthe -lactam carbonyl carbon atom was eliminated. Consequently,the kcat values for hydrolysis ofbenzylpenicillin and nitrocefin have been reduced by104-105 compared with the wild-typeenzyme.The crystal structure of S70A -lactamase has been determined at2.1 Å resolution. With the exceptionof the mutation site, the structure is identical to that of the nativeenzyme. The residual activity is attributedeither to mistranslation that leads to production of wild-type enzymeand/or to remaining features of theactive site that stabilize the tetrahedral transition state.Soaking of the crystals with ampicillin orclavulanate,followed by flash-freezing, has been carried out and the structuresexamined at 2.0 Å resolution. Forboth experiments, the difference electron density maps revealed buildupof density in the active site thatpresumably corresponds to -lactam binding. However, neitherelectron density is sufficiently clear fordefining the atomic details of the bound compounds. The K73H-lactamase has been prepared to testthe possible role of Lys73 in proton transfer. It exhibits nodetectable activity toward benzylpenicillin,and 105-fold reduction of kcat fornitrocefin hydrolysis compared with the wild-type enzyme. Nosignificantrecovery of activity has been measured when the pH was varied between5.0 and 8.0. The crystal structureof K73H -lactamase has been determined at 1.9 Å resolution.While the overall structure is similar tothat of the native enzyme, the electrostatic interactions between His73and neighboring residues indicatethat the imidazole ring is positively charged. In addition, thehydroxyl group of Ser70 adopts a positionthat is incompatible with nucleophilic attack on substrates. Acrystal soaked with ampicillin was flash-frozen, and diffraction data were collected at 2.1 Å resolution.The electron density map showed noindication of substrate binding.