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

对在三磷酸腺苷和镁离子存在下催化γ-L-谷氨酸-L-半胱氨酸和甘氨酸合成谷胱甘肽的大肠杆菌B菌株谷胱甘肽合成酶的X-射线分析表明，该酶的Ile226-Gly241具有电子密度图上看不到的柔性环状结构。推测该环可能存在于底物结合部位附近，并在酶反应中发挥重要作用。然后，通过精氨酸内肽酶对柔性环的特异性切割、突变分析和化学修饰，对柔性环在反应中的作用进行了结构研究。结果表明，柔性环对于底物结合和酶反应都是必不可少的。三磷酸腺苷和酰基磷酸中间体中的高能键对亲核攻击非常敏感。如果水分子与三磷酸腺苷反应，而不是与适当的反应物反应，三磷酸腺苷就会被水水解，然后酶反应就不能正确地进行到更好的产物。柔性环可以保护中间体或酶-底物复合体，使其不受通过中间体或复合体与水的相互作用而发生的不良反应的影响。当底物与酶结合时，柔性环似乎通过改变底物的构象来缠绕它们的结合部位来保护底物免受水的任何攻击。该环在谷胱甘肽合成酶中的功能似乎类似于磷酸丙糖异构酶。有关该酶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.