Structural studies on relationship between the flexibility of loop and the function of enzyme

对大肠杆菌B的谷胱甘肽合成酶进行了X射线分析，该酶在ATP和镁离子存在下催化γ-L-Glu-L-Cys和Gly合成谷胱甘肽，结果表明该酶的Ile 226-Gly 241具有在电子密度图中不可见的柔性环结构。该环可能存在于底物结合位点附近，在酶促反应中起重要作用。然后，结构的灵活的环在反应中的作用进行了研究，使用特异性切割的环由β-淀粉酰内肽酶，突变分析和化学修饰。事实证明，柔性环对于底物结合以及酶反应是必不可少的。ATP和酰基磷酸酯中间体中的高能键对亲核攻击相当敏感。如果水分子与ATP而不是适当的反应物反应，则ATP被水水解，然后酶促反应不能朝着优选的产物适当地进行。柔性环可以保护中间体或酶-底物复合物免受可能通过中间体或复合物与水的相互作用而发生的不期望的反应。当底物与酶结合时，柔性环似乎通过改变其构象以缠绕它们的结合位点来保护底物免受任何水的攻击。谷胱甘肽合成酶中的环的功能似乎与磷酸丙糖异构酶中的类似。酶的反应机理的详细信息将通过使用同步辐射的时间分辨劳厄实验获得。

X-ray analysis of glutathione synthetase from Escherichia coli B which catalyzes the synthesis of glutathione from gamma-L-Glu-L-Cys and Gly in the presence of ATP and magnesium ion revealed that Ile226-Gly241 of the enzyme had an flexible loop structure which was unvisible in the electron density map. It was expected that this loop may exist near the substrate binding sites and play an important role in the enzymatic reaction. Then, structural studies on the role of the flexible loop in the reaction were carried out using specific cleavage of the loop by arginylendopeptidase, mutational analysis and chemical modification. It turned out that the flexible loop is essential to the substrate binding as well as the enzyme reaction. The high-energy bonds in ATP and acylphosphate intermediate are quite sensitive against nucleophilic attack. If a water molecule reacts with ATP instead of a proper reactant, ATP ishydrolyzed by the water and then the enzymatic reaction does not proceed properly toward the preferable product. The flexible loop may protect the intermediate or the enzyme-substrate complex from undesirable reactions which may take place through interaction of the intermediate or the complexes with water. When the substrates bind to the enzyme, the flexible loop seems to protect the substrates from any attack of water by changing its conformation to wrape their binding sites. The function of the loop in glutathione synthetase seems to be similar to that in triosephosphate isomerase. Detailed information on the reaction mechanism of the enzyme will be obtained by time-resolved Laue experiments using synchrotron radiation.