分子动力学模拟研究CGTase酶活性区域的热稳定性

Insight into thermal stability of CGTase active site region by molecular dynamics simulation

环糊精葡萄糖基转移酶(cyclodextrin glycosyltransferase,CGTase,E.C.2.4.1.19)是食品、医药和化妆品等领域中的重要酶。文中通过分子动力学模拟研究了CGTase活性位点区域中分子内非共价键相互作用对蛋白质热稳定性的影响,以及在热压力下这些分子内非共价键相互作用又会如何发生变化。研究发现:在CGTase酶活性区域中分布着较多的盐桥和氢键网络体系,分析盐桥间的距离变化发现这些盐桥对高温具有很好的抗性,同时,高温下盐桥网络又比单个盐桥更加稳定;对该区域中氢键分析,发现随着模拟温度的升高,带电荷氨基酸之间形成的氢键在高温下具有很好的稳定性,同样,高温下氢键网络也比单个氢键更加稳定;通过对非共价键相互作用的分析中发现活性位点两侧的氨基酸残基与酶的热稳定性也有很大的关系。该蛋白酶的热稳定性与非共价键连接的数量、存在形式及形成非共价键相互作用的氨基酸空间位置都息息相关。该结果为用酶工程设计改造环糊精葡萄糖基转移酶热稳定性提供了突变方向,同时,为理解酶蛋白的热稳定机制,结构与功能关系的研究提供有益参考。

Cyclodextrin glucanotransferase （CGTase, EC 2.4.1.19） is an important enzyme in food, pharmaceutical and cosmetic, etc. The objective of this study is to investigate the contribution of non-covalent intramolecular interactions to protein CGTase stability and how changes in these interactions in response to thermal stress. The results show： there are more salt bridge and hydrogen bond network system which distribute in the CGTase enzyme active area. Salt bridge analyses reveals that in one hand, salt bridges involved in active site region are relatively stong at elevated temperature; in the other hand, salt bridge network is more stable than the single salt bridge. Hydrogen bond analyses reveals that the hydrogen bond formed between the charged amino acid has good stability at high temperature, and, hydrogen bond network is more stable than the single hydrogen bond also. Meanwhile, the amino acids, beside the active site, are contributed to the protein thermostability also. These results suggest the protein CGTase stability is related to the number, existent form and amino acid spatial position of the formation of non-covalent interaction. The work provides the direction for mutation to improve enzymatic thermosmbility, furthermore, a useful reference for understanding the mechanism of the thermal stability and structure-fimction relationship.