Capturing Transit Structures by Kinetic Crystallography

为了研究功能蛋白质（如酶）的作用，必须直接观察它们的运动结构。在这项研究中，我们的目标是捕捉短期生产性米氏复合物的结构在谷胱甘肽合成酶的反应。为了实现这一点，我们使用了一个过渡态类似物抑制剂（TSA），这是受到酶催化磷酸化的酶活性位点内的ATP作为底物的酶。因此，我们使与这种抑制剂和笼状ATP复合的酶结晶，以防止磷酸化。该系统允许在光裂解时释放ATP以引发酶活性位点内的抑制剂的磷酸化。我们使用白色光束劳厄衍射法或传统的单色法与闪冷。由于劳厄衍射法存在低分辨数据完整性差和X射线对晶体损伤大的缺点，我们计划用单色法来补偿劳厄衍射法的结果。在欧洲同步辐射装置（ESRF）的ID9光束线上，我们成功地测量了非常好的劳厄衍射数据。我们还存档安装ESRF开发的计算机程序，以在京都大学化学研究所的实验室中处理劳厄衍射。结果表明，我们已经捕捉到的结构的TSA未磷酸化后立即光解。我们还确定了时间分辨的电影在TSA被磷酸化与ATP在酶的活性位点。

To investigate the action of functional proteins such as enzymes, it is essential to visualize their structure in motion directly. In this studies, we aim to capture the short-lived productive Michaelis complex structure during the reaction of glutathione synthetase. To accomplish this, we used a transition-state analogue inhibitor (TSA) which is subjected to enzyme-catalyzed phosphorylation by ATP within the enzyme active site as a substrate of enzyme. We thus crystallized the enzyme complexed with this inhibitor and a caged-ATP to prevent the phosphorylation. This system allows to release ATP upon photdlysis to initiate the phosphorylation of the inhibitor within the enzyme active site. We used white beam Laue diffraction method or conventional monochromatic methods with flash cooling. We planed to use the monochromatic methods to compensate the results of Laue methods since the Laue diffraction has two disadvantages, low completeness of the low resolution data and high X-ray damage with crystals. We succeed to measure very good Laue diffraction data at the beamline ID9 at European Synchrotron Radiation Facility (ESRF). We also archived to install the computer program developed at ESRF to handle the Laue diffraction in our laboratory at Institute for Chemical Research, Kyoto University. The results showed that we have captured the structure of TSA unphosphorylated at immediately after photolysis. We also determine time-resolved movies during the TSA is being phosphorylated with ATP at the enzyme active site.