天花粉蛋白Glu160在N-糖苷酶活性中的作用

用浸泡法得到了（Ｅ１６０Ａ）天花粉蛋白（ｔｒｉｃｈｏｓａｎｔｈｉｎ， ＴＣＳ），（Ｅ１６０Ｄ）ＴＣＳ与Ａｄｅ 和（Ｅ１６０Ａ）ＴＣＳ与ＦＭＰ复合物的晶体．在Ｍａｒ Ｒｅｓｅａｒｃｈ 面探测器系统上分别收集了０．２０ ｎｍ ，０．１９ｎｍ 和０．２０５ ｎｍ 分辨率的Ｘ 射线衍射数据，数据处理用Ｍａｒ Ｓｃａｌｅ 程序系统完成．用同晶差值Ｆｏｕｒｉｅｒ法解析了（Ｅ１６０Ａ）ＴＣＳ－Ａｄｅ，（Ｅ１６０Ｄ）ＴＣＳ－Ａｄｅ 和（Ｅ１６０Ａ）ＴＣＳ－ＦＭＰ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０．１６６，０．１７６，０．１７９．键长和键角的ＲＭＳ偏差分别为０．００１０ ｎｍ 和２．５０３°，０．００１３ ｎｍ 和２．６６５°，０．００１２ ｎｍ 和２．６７６°．在这三个结构中均未见到Ｇｌｕ１８９侧链方向的改变．Ａｄｅ 或ＦＭＰ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分别突变成Ａｌａ 和Ａｓｐ，仍能与ＡＭＰ发生Ｎ－糖苷酶反应，但是活性降低了一些．可见Ｇｌｕ１６０对ＴＣＳ与ＡＭＰ的作用是重要的，但不是必要的．

Role of Glu160 in the N-glycosidase Activity of Trichosanthin

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.