外源性苯甲酰牙子碱代谢酶的首次发现与合理化设计及其催化机制研究

苯甲酰芽子碱（BZE）是可卡因的主要有毒代谢物，体内半衰期长，主要负责可卡因的长期毒性。内源性丁酰胆碱酯酶是目前唯一报道的BZE代谢酶（BZEase），可降解BZE为无生物活性的分子，但催化活性低、糖基化修饰复杂、重组表达困难。因此发现和改造新型BZEase，实现催化活性高、表达经济高效，对开发可卡因酶法解毒治疗具有重要价值。课题组首次发现细菌来源且能高效表达的可卡因酯酶（CocE）能催化BZE苯酯水解，初步改造获得V116K突变体，对BZE的催化效率（kcat/KM）提高了64倍。本项目拟采用结构生物学和计算机模拟方法，构建准确的BZEase-BZE结合模型，深入阐明BZEase的催化机理，并确定影响限速步过渡态结构稳定性的因素。继而运用计算机辅助设计和实验验证相结合的策略，进一步合理化设计改造BZEase，以获得更高活性BZEase突变体，为可卡因的彻底解毒治疗提供新的候选药物。

Benzoylecgonine (BZE) is the major toxic metabolite of cocaine, and is responsible for the long-term cocaine-induced toxicity due to its long residence time in human. Endogenous butyrylcholinesterase is currently recognized as the only BZE-metabolizing enzyme (BZEase), which can degrade BZE into biologically inactive metabolites. However, its low catalytic activity and the great challenge to efficiently and economically produce recombinant BChE limit its potential application as an BZEase. Hence, it is critical to discover new BZE-metabolizing enzymes for treatment of cocaine toxicity, an effort to identify new promising biopharmaceuticals that are highly active in BZE hydrolysis and cost-effective in production. Our preliminary data demonstrated for the first time that bacterial cocaine esterase (CocE) can catalyze the hydrolysis of BZE at benzoyl ester group. However, the catalytic efficiency of wild-type CocE against BZE is too low to effectively metabolize BZE to completely eliminate its toxicity. The encouraging mutant V116K we discovered in the first round showed 64-fold improved catalytic efficiency（kcat/KM）, which demonstrates the feasibility of our approach. Therefore, the proposed investigation is intended to build an accurate BZEase-BZE binding model, uncover the detailed mechanism for BZEase-catalyzed hydrolysis of BZE, and determine the factors that could affect the stability of the rate-determining transition state of the enzymatic reaction. In light of the aforementioned insights, high-activity mutants of BZEase could be rationally designed and discovered using novel integrated computational-experimental approach. An effort to discover new BZEase mutants with highly improved catalytic activity against BZE could led to the discovery of potential therapeutic agents for complete elimination of cocaine’s toxicity.