尿苷二磷酸葡糖醛酸转移酶催化尼古丁及其主要代谢物的葡糖醛酸化反应的理论研究

Nicotine and its primary metabolites cotinine and trans-3’-hydroxycotinine (3HC) have been found to be harmful and closely related with tobacco addiction. It has been reported that uridine diphosphate glucuronic acid transferase UGT2B10 and UGT2B17 can catalyze the glucuronidation of nicotine, cotinine and 3HC, and transform them to the nontoxic glucuronides, indicating the potential role of UGT2B10 and UGT2B17 as nicotine antidotes. In this project, the detailed reaction mechanisms as well as the minimum-energy reaction pathway and the rate-determining transition state structures for UGT2B10/UGT2B17-catalyzed glucuronidation of nicotine, cotinine and 3HC will be fully explored by employing a series of computational methods such as homology modeling, molecular docking, molecular dynamics (MD) simulations and quantum mechanical/molecular mechanical (QM/MM) calculations. Followed by the reaction mechanism calculations, the important factors contributing to accelerate the UGT2B10/UGT2B17-catalyzed glucuronidation reactions will be identified. Then, in order to improve the performance of UGT2B10/UGT2B17 enzymes, new mutations aim to stabilize the rate-determining transition-states will be introduced and further confirmed by performing MD simulations and QM/MM calculations. This project would provide some useful information for rational engineering of more efficient UGT2B10/UGT2B17 mutants which not only can help accelerate the glucuronidation of nicotine and its primary toxic metabolites, but also could be used as potential therapeutic candidates to treat tobacco addiction.

尼古丁及其在体内的主要代谢物可替宁和反式3′-羟基可替宁(3HC)对人体都有害且与烟瘾形成密切相关。尿苷二磷酸葡糖醛酸转移酶UGT2B10和UGT2B17能把尼古丁及其主要代谢物转化为无毒的葡糖醛酸化合物，是潜在的尼古丁解毒剂。本项目拟用同源模建、分子对接、分子动力学模拟和量子力学与分子力学相结合的方法对UGT2B10/UGT2B17催化尼古丁、可替宁和3HC的葡糖醛酸化反应进行理论研究，确定能量最低反应路径和决速步骤过渡态的结构，分析能加速酶催化反应的关键因素，再以稳定决速步骤过渡态为导向，借助理论计算设计一些突变以提高UGT2B10/UGT2B17的活性，为人们合理设计高活性UGT2B10/UGT2B17突变酶催化尼古丁及其主要代谢物的葡糖醛酸化提供理论参考。基于高活性UGT突变酶设计的医用药物不但有助于烟民体内的尼古丁及其主要有毒代谢物快速安全地排出体外，还有望用于治疗烟瘾。

尼古丁及其在体内的主要代谢物可替宁和反式3′-羟基可替宁(3HC)对人体都有害且与烟瘾形成密切相关。尿苷二磷酸葡糖醛酸转移酶UGT2B10和UGT2B17能把尼古丁及其主要代谢物转化为无毒的葡糖醛酸化合物，是潜在的尼古丁解毒剂。在本项目中，我们用同源模建、分子对接、分子动力学模拟和量子力学与分子力学相结合的方法对UGT2B10/UGT2B17催化尼古丁、可替宁和3HC的葡糖醛酸化反应进行理论研究，确定能量最低反应路径和决速步骤过渡态的结构，分析能加速酶催化反应的关键因素，再以稳定决速步骤过渡态为导向，借助理论计算设计一些突变以提高UGT2B10/UGT2B17的活性，为人们合理设计高活性UGT2B10/UGT2B17突变酶催化尼古丁及其主要代谢物的葡糖醛酸化提供理论参考。分子动力学模拟计算表明SRH形态的尼古丁是UGT2B10的主要反应底物，QM/MM反应机理研究表明UGT2B10催化SRH形态的尼古丁发生葡糖醛酸化反应遵循SN2协同反应机制，N-C成键的同时C-O键断裂。计算所得的反应能垒是25.1 kcal/mol，这与根据实验动力学数据推算的活化自由能垒（24.2~24.9 kcal/mol）非常接近。同时，我们的理论计算也表明SRH形态的可替宁是UGT2B10的主要反应底物，UGT2B10代谢可替宁的葡糖醛酸化反应也遵循SN2协同反应机制，N-C成键的同时C-O键断裂。计算所得的反应能垒是26.5 kcal/mol，这与根据实验动力学数据推算的活化自由能垒（25.8 kcal/mol）非常接近。得到的反应机制不但对于在分子水平上理解UGT酶催化含氮化合物的N-葡糖醛酸化反应过程有帮助，而且能为将来合理设计高效的UGT2B10突变酶加速尼古丁类化合物的代谢提供参考.