Fast crystallography of enzymatic readim in crystals
Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants|
|Research Institution||KYOTO UNIVERSITY|
ODA Junichi Kyoto University, Institute for Chemical Research Professor, 化学研究所, 教授 (50027041)
KATO Hiroaki Kyoto University, Institute for Chemical Research Assistant, 化学研究所, 助手 (90204487)
SCHLICHING Ilme Max Planck Institute Senior Investigator, 主任研究員
PETSKO Gregory a. Brandeis University, Basic Medical Sciences Research Center Professor, 基礎医科学研究所, 教授
YAMAGUCHI Hiroshi Osaka University, Institute for Protein Research Assistant, 蛋白質研究所, 助手 (10252719)
NISHIOKA Takaaki Kyoto University, Institute for Chemical Research Associate Professor, 化学研究所, 助教授 (80026559)
ANDERSSON Inger Swedish University of Agricultural Sciences, Uppsala Biomedical Center Doctor Re, ウプサラ生医学センター, 博士研究員
HAJDU Janos Oxtord University, Laboratory of Molecular Biophysics Lecturer, 分子生物物理学研究室, 講師
|Project Period (FY)
Completed(Fiscal Year 1995)
|Budget Amount *help
¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1995 : ¥2,500,000 (Direct Cost : ¥2,500,000)
|Keywords||X-ray crystallography / Kinetic crystallography / Protein Crystallography / cryo-crystalloqraphy / glutathione synthetase / enzym reaction in crystals|
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.
Research Output (6results)