Capturing Transit Structures by Kinetic Crystallography

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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.