天花粉蛋白Glu189在N-糖苷酶活性中的作用

Role of Glu189 in the N- glycosidase Activity of Trichosanthin

培养了(E160A,E189A)TCS(天花粉蛋白)的单晶。用浸泡法得到了(E160A,E189A)TCS与Ade复合物的晶体。在MarResearch面探测器系统上分别收集了均为0.20nm分辨率的X射线衍射数据,数据处理用MarScale程序系统完成。用同晶差值Fourier法解析了(E160A,E189A)TCS和(E160A,E189A)TCS-Ade的晶体结构,结构修正利用X-PLOR程序.修正结果,晶体学R因子分别为0.180、0.184,键长和键角的RMS偏差分别为0.0012nm和2.566°、0.0012nm和2.622°。在(E160A,E189A)TCS-Ade中,Ade仍结合在N-糖苷酶活性口袋之中,它夹在Tyr70和Tyr111两个侧链环之间,与Tyr70环近乎平行。这一结果表明:TCS中的Glu160和Glu189同时突变成Ala,仍能与AMP发生N-糖苷酶反应.前文已经证明在(E160A)TCS中Glu189没有援救作用。目前,没有发现Glu189对TCS与AMP的直接作用,但Glu189与其它残基的协同作用及其在TCS与rRNA作用中扮演什么角色,尚待进一步研究。

Crystals of (E160A)TCS Ade complex,(E160D)TCS Ade complex and (E160A)TCS FMP complex were obtained by soaking (E160A)TCS and (E160D)TCS in artificial mother liquor containing 10 mg/ml AMP or FMP for 48 hours. X ray diffraction data were collected to 0 20 nm , 0 19 nm and 0 205 nm resolution on a Mar Research area detector. The Mar Scale program was applied to data processing. Difference Fourier Method was applied to the structure analysis and X PLOR package used for their refinement. The final crystallographic R factor was 0 166,0 176 and 0 179 respectively. The RMS deviation of bond length and angle of the mutant and complex were 0 0010 nm and 2 503°, 0 0013 nm and 2 665°, 0 0012 nm and 2 676° respectively. The direction change of Glu 189 side chain was not observed in these crystal structures. Ade or FMP bound to proteins at the proposed active region with its adenine ring stacking between Tyr70 and Tyr111. According to the above results, when Glu160 of TCS was substituted respectively by Ala and Asp, it could interact with AMP. But the activities of (E160A)TCS and (E160D)TCS were less than TCS. As a result the role of Glu160 was important in the interaction between TCS and AMP, but not essential.