Capturing Transit Structures by Kinetic Crystallography

为了研究酶等功能性蛋白质的作用，有必要直接观察它们的运动结构。在本研究中，我们的目标是捕获在谷胱甘肽合成酶反应过程中的短寿命生产Michaelis复合体结构。为了实现这一目标，我们使用了一种过渡态类似抑制剂（TSA），该抑制剂作为酶的底物，在酶活性位点内被ATP催化磷酸化。因此，我们结晶了与该抑制剂和笼状atp络合的酶，以防止磷酸化。该系统允许在光解时释放ATP，以启动酶活性位点内抑制剂的磷酸化。我们采用白光劳厄衍射法或传统的单色法进行闪冷。我们计划用单色法来补偿Laue法的结果，因为Laue衍射有两个缺点，低分辨率数据的完整性低，x射线对晶体的损伤大。我们成功地在欧洲同步辐射装置（ESRF）的光束线ID9上测量了非常好的劳厄衍射数据。我们还存档了在京都大学化学研究所的实验室中安装ESRF开发的计算机程序来处理劳厄衍射。结果表明，我们捕获了光解后立即未磷酸化的TSA结构。我们还测定了TSA在酶活性位点被ATP磷酸化时的时间分辨电影。

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.