半理性设计提高丁酰胆碱酯酶催化生长激素释放肽活性

Semi-rational design improves the catalytic activity of butyrylcholinesterase against ghrelin

摘要:

生长激素释放肽(ghrelin)是一种对于生长激素的分泌、食欲等具有重要调节作用的脑肠肽，还可调节体内糖脂代谢过程，已成为相关疾病治疗的研究热点。人体中的丁酰胆碱酯酶(human butyrylcholinesterase, hBChE)可将ghrelin水解为去酰基化状态，但是催化效率极低，限制了其应用。本研究通过HotSpot Wizard 3.0分析原核可溶性表达的hBChE突变体结构，理性选取10个新突变体，再对不同底物催化动力学与热力学稳定性进行测定，最终筛选出的新突变体E197D与A199S对于ghrelin催化水解活性分别上升4.6倍与3.5倍，为实现体外给药调节体内ghrelin进行相关疾病治疗提供了可能。

Abstract:

Ghrelin, a hormone mainly produced and released by the stomach, has numerous functions, including releasing growth hormones, regulating appetite, and processing sugar and lipids. Researchers have made great efforts to study the relationship between ghrelin and metabolic diseases. It is believed that human butyrylcholinesterase (hBChE) could hydrolyze ghrelin to the inactive form (desacyl-ghrelin). However, the low catalytic activity of wild hBChE against ghrelin hinders the clinical application. Recently, a soluble catalytically active hBChE mutant was successfully expressed in Escherichia coli for the first time. We then adopted HotSpot Wizard 3.0 to analyze the mutant structure and rationally selected 10 mutants. Furthermore, we determined the catalytic activities of the mutants against several substrates and the thermostability of these mutants. The results showed that the mutants E197D and A199S improved catalytic activity against ghrelin by 4.6 times and 3.5 times, respectively. The findings provide clues for treating endocrine diseases with the agents for regulating ghrelin.