Redesign of Butyrylcholinesterase for Cocaine Metabolism

描述（由申请人提供）：本项目主要研究人类丁基胆碱酯酶（BChE）的合理再设计，以加速人体可卡因的代谢。通过给药BChE促进可卡因代谢已被认为是一种可能的治疗可卡因过量和成瘾的策略。然而，这种血浆酶对天然存在的(-)-可卡因的催化活性比对相对无生物活性的(+)-可卡因异构体的催化活性低三个数量级。（+）-可卡因在进入中枢神经系统前几秒钟就能从血浆中清除。该项目的长期目标是指导我们的合作者使用位点定向诱变技术构建BChE突变体，以便该突变体可以作为人体内的外源酶，其对(-)-可卡因的催化活性可与野生型BChE对(+)-可卡因的催化活性相媲美。为此，将使用最先进的计算技术来揭示(-)-可卡因和(+)-可卡因之间酶反应的机制差异，并为具有(-)-可卡因活性的BChE突变体的理论设计建立合理的基础。本建议所涉调查的具体目的包括：1。确定BChE与(-)-可卡因和(+)-可卡因结合的结构和动力学，包括非预反应性和预反应性BChE-底物复合物。2. 通过在适当选择的模型体系上进行反应坐标计算，揭示bche催化(-)-可卡因和(+)-可卡因在苯甲酰酯上水解的基本反应途径；3. 通过对酶-反应物、过渡态、中间体和产物配合物进行MD模拟，研究酶解过程中BChE与(-)-可卡因和(+)-可卡因结合的动力学和变化；4. 通过量子力学和自由能摄动计算（QM-FE）来评估酶解的自由能分布和动力学；5. 基于从Specific Aim 2获得的结合信息和从Specific Aim 3获得的能量信息，设计对(-)-可卡因具有更高活性的BChE突变体。

DESCRIPTION (provided by applicant): This project focuses on the rational redesign of human butyrylcholinesterase (BChE) in order to accelerate cocaine metabolism in man. Enhancing cocaine metabolism by administration of BChE has been recognized as a possible treatment strategy for cocaine overdose and addiction. However, the catalytic activity of this plasma enzyme is three orders-of-magnitude lower against the naturally occurring (-)-cocaine than that against the relatively biologically inactive (+)-cocaine isomer. (+)-cocaine can be cleared from plasma in seconds prior to partitioning into the central nervous system. The long-term goal of this project is to guide the construction of a BChE mutant using site-directed mutagenesis by our collaborators so that the mutant can be used as an exogenous enzyme in human body with a catalytic activity for (-)-cocaine comparable to that of wild-type BChE for (+)-cocaine. For this purpose, state-of-the-art computational techniques will be used to uncover the mechanistic difference of the enzymatic reaction between (-)-cocaine and (+)-cocaine and to establish a rational basis for theoretical design of BChE mutants with an improved activity for (-)-cocaine. The specific aims of the investigation involved in the present proposal include: 1. To determine structures and dynamics of the BChE binding with (-)-cocaine and (+)-cocaine, including the non-prereactive and prereactive BChE-substrate complexes. 2. To uncover fundamental reaction pathways for BChE-catalyzed hydrolysis of (-)-cocaine and (+)-cocaine at the benzoyl ester by performing reaction coordinate calculations on properly chosen model systems; 3. To investigate dynamics and changes of BChE binding with (-)-cocaine and (+)-cocaine during the enzymatic hydrolysis by carrying out MD simulations on the enzyme-reactant, transition state, intermediate and product complexes; 4. To evaluate free energy profiles and kinetics of the enzymatic hydrolysis by performing combined quantum mechanical and free energy perturbation calculations (QM-FE); 5. To design BChE mutants expected to have higher activity for (-)-cocaine based on the binding information obtained from Specific Aim 2 and the energetic information from Specific Aim 3.