Fast crystallography of enzymatic readim in crystals

本研究的目的是建立一个系统，使我们能够通过晶体学捕捉酶的动态结构。为此，我们对结晶状态下的酶促反应进行了动力学分析，并开发了一些快速x射线衍射强度测量和低温结晶学技术。我们之所以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，而且这种酶能够催化另一种简单的反应，在这种反应中，过渡态类似物被ATP磷酸化。由于底物向晶体的扩散速度比晶体状态下的酶促反应速度要慢，我们使用笼型atp来同步反应。通过氙灯光解触发快速释放笼中atp。晶体中的磷酸化反应比溶液中的慢，在20℃时其半衰期为3分钟。结果表明，当我们收集几秒钟的x射线数据时，我们所瞄准的过境结构是可测量的。我们在日本筑波高能物理研究所光子工厂用同步辐射和Weissenberg照相机采集了20℃时的x射线数据。由于数据收集需要1小时，所以我们捕捉到的结构就是磷酸化反应完成的结构。然而，该结构使我们发现，即使在结晶状态下，酶催化的磷酸化反应也是进行的，其结构与溶液中得到的结构相同。为了降低反应速率，我们在系统中应用了低温结晶技术。我们发现即使在160°C的温度下，晶体复合物也能存活，并测量了x射线数据。我们开发的技术可以用于其他酶晶体。现在，我们正试图找到在光解后快速冷却的方法。

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.