WT-L1与Co(Ⅱ)-L1的光谱表征及催化水解β-内酰胺类抗生素的动力学研究  

Spectroscopic characterization of WT-L1 and Co(Ⅱ)-L1 and their kinetic studies on catalyzing β-lactam antibiotics hydrolysis

作　　者：闫小艳[1] 高会洲[1] 冯吉利[1] 杨霞[1] 杨科武[1] 

机构地区：[1]合成与天然功能分子化学教育部重点实验室西北大学化学与材料科学学院,西安710069

出　　处：《中国抗生素杂志》2011年第5期388-393,共6页Chinese Journal of Antibiotics

基　　金：国家自然科学基金(20972127);教育部博士点基金(200806970005);人力资源部基金(SLZ2008013);陕西省自然科学基金(2009JM2002);陕西省教育厅基金(09JK786)

摘　　要：目的表征金属β-内酰胺酶(MBL)L1的结构和动力学特性,以其发展MBL的通用型抑制剂。方法在大肠埃希菌BL21(DE3)中表达并纯化野生型L1(Wild type L1,WT-L1),以SDS-PAGE电泳确认;用EDTA除去WT-L1中的Zn(Ⅱ)离子得Apo-L1,再用Co(II)滴定制得Co(Ⅱ)-L1;通过UV-Vis,CD及荧光光谱表征WT-L1及Co(Ⅱ)-L1活性中心的结构;采用稳态动力学方法测定WT-L1及Co(Ⅱ)-L1催化3类9种β-内酰胺类抗生素水解反应的酶动力学参数,确定抗生素对L1的稳定性。结果获得纯化的WT-L1和重组的Co(Ⅱ)-L1;光谱表征表明L1具有两个金属活性中心,Co(II)-L1的二级结构发生显著变化;WT-L1对碳青霉烯类抗生素的酶促活性最强,青霉素类次之,头孢类抗生素较弱,Co(Ⅱ)-L1比WT-L1的酶催活性弱。结论 Co(Ⅱ)-L1的重组实现了对MBL-L1的结构表征,L1对青霉素类抗生素呈现强的催化活性,对头孢类抗生素稳定。Objective In order to develop universal inhibitors of metallo-β-1actamases（MBLs）, the structure and kinetic properties of MBL-L 1 are characterized. Methods The wild type L 1 （WT-L 1） is expressed in BL21 （DE3） E. coli and purified and confirmed by SDS-PAGE. Co（II）-L1 is prepared by addition of Co（II） to apo-L1, which is obtained by removing Zn（II） in it with EDTA. The active site structures of WT-L1 and Co（II）-L1 are characterized by UV-Vis, CD and fluorescence spectrum. With steady-state kinetics, the kinetic data of 9 kinds of β-lactam antibiotics hydrolysis catalyzed by WT-L1 and Co（II）-L1, respectively, are determined, also, the stability of the antibiotics to L1 are evaluated. Results The purified WT-L1 and Co（II）-L1 were obtained, the results of spectroscopic characterization indicated that the L1 has two metal centers, and compared with apo-L1, the secondary structure of Co（II）-L1 changed clearly. WT-L1 on the enzymatic carbapenem most active, followed by penicillins, cephalosporins weak, comparing the catalytic activity, Co（II）-L1 is weaker than WT-L1. Conclusion Recombinant of Co（II）-L1 makes it possible to characterize MBL-L1- L1 possesses strong activity for penicillin antibiotics and has no activity for cephalosporin antibiotics.

关 键 词：金属β-内酰胺酶L1 金属酶重组 光谱表征 β-内酰胺类抗生素:酶催化动力学 

分 类 号：R978.11[医药卫生—药品]