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Fast crystallography of enzymatic readim in crystals

晶体中酶促再结晶的快速晶体学

基本信息

批准号：
07044195
负责人：
ODA Junichi
金额：
$ 1.6万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1995
资助国家：
日本
起止时间：
1995 至无数据
项目状态：
已结题

项目摘要

The purpose of this study is construction of a system which allow us to catch the dynamic structure of enzymes in action by crystallography. For this purpose, we did the kinetic analysis of enzymatic reaction in crystalline state, and the development of some techniques for fast X-ray diffraction intensity measurement and cryo-crystallography. We chose Escherichia coli glutathione synthetase for this study because we already solved its crystal structure and the enzyme is able to catalyze another simple reaction in which a transition-state analogue is phosporylated with ATP.As the diffusion of substrate into a crystal should slower than the enzymatic reaction rate in crystalline state, we used caged-ATP for the synchronization of the reaction. The caged-ATP was uncaged rapidly triggered by Xenon flash lamp photolysis. The phosphorylation reaction in the crystal was slower than that in solution and its half life was 3 minutes at-20ﾟC.The results indicate that the transit structure we aimed is measurable when we collect X-ray data for several seconds.We collected the X-ray data at-20ﾟC using synchrotron radiation and Weissenberg camera at Photon Factory, Institute for High Energy Physics, Tsukuba. Since the data collection needs 1 hour, the structure we caught was the structure in which the phosphorylation reaction is finished. However, the structure allow us to find that the phosphorylation reaction catalyzed by the enzyme is progress even in the crystalline state and it is the same structure as that obtained in solution.In order to slow down the reaction rate, we applied cryo-crystallographic technique to our system. We found the condition in which the complex the crystals survived even at-160ﾟC and did to measure the X-ray data. The technique we developed was possible to utilize for the other enzyme crystals. Now, we are trying to find the way of flash cooling soon after the photolysis.

这项研究的目的是构建一个系统，使我们能够通过结晶学捕捉作用中的酶的动态结构。为此，我们对结晶状态下的酶反应进行了动力学分析，并发展了一些快速X射线衍射强度测量和低温结晶学技术。我们选择大肠杆菌谷胱甘肽合成酶进行这项研究，是因为我们已经解决了它的晶体结构，并且该酶能够催化另一种简单的反应，在该反应中，过渡态类似物与ATP发生磷酸化反应。由于底物在晶体中的扩散速度应该慢于结晶状态下的酶反应速度，因此我们使用笼式ATP来同步反应。被氙灯光解快速触发的笼状三磷酸腺苷。晶体中的磷酸化反应比溶液中的反应慢，在-20ﾟC下的半衰期为3分钟。结果表明，我们目标的跃迁结构在收集几秒钟的X射线数据时是可测量的。我们在筑波高能物理研究所的光子工厂使用同步辐射和魏森伯格相机收集了-20ﾟC下的X射线数据。由于数据采集需要1小时，所以我们捕捉到的结构是完成磷酸化反应的结构。然而，该酶的结构使我们发现，即使在结晶状态下，该酶催化的磷酸化反应也是进行的，并且它与在溶液中得到的结构相同。为了减缓反应速度，我们将冷冻结晶技术应用到我们的体系中。我们找到了晶体在-160ﾟC下仍能存活的条件，并进行了X射线数据测量。我们开发的技术也有可能用于其他酶晶体。现在，我们正在努力寻找在光解后不久进行闪冷的方法。