Yang Wenchao, Pan Yongmei, Zheng Fang, Cho Hoon, Tai Hsin-Hsiung, Zhan Chang-Guo
Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, PR China.
Biophys J. 2009 Mar 4;96(5):1931-8. doi: 10.1016/j.bpj.2008.11.051.
It is recognized that an ideal anti-cocaine treatment is to accelerate cocaine metabolism by producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., butyrylcholinesterase (BChE)-catalyzed hydrolysis of cocaine. BChE mutants with a higher catalytic activity against (-)-cocaine are highly desired for use as an exogenous enzyme in humans. To develop a rational design for high-activity mutants, we carried out free-energy perturbation (FEP) simulations on various mutations of the transition-state structures in addition to the corresponding free-enzyme structures by using an extended FEP procedure. The FEP simulations on the mutations of both the free-enzyme and transition-state structures allowed us to calculate the mutation-caused shift of the free-energy change from the free enzyme (BChE) to the transition state, and thus to theoretically predict the mutation-caused shift of the catalytic efficiency (k(cat)/K(M)). The computational predictions are supported by the kinetic data obtained from the wet experiments, demonstrating that the FEP-based computational design approach is promising for rational design of high-activity mutants of an enzyme. One of the BChE mutants designed and discovered in this study has an approximately 1800-fold improved catalytic efficiency against (-)-cocaine compared to wild-type BChE. The high-activity mutant may be therapeutically valuable.
人们认识到，理想的抗可卡因治疗方法是通过类似于可卡因主要代谢途径的方式产生无生物活性的代谢产物来加速可卡因代谢，即丁酰胆碱酯酶（BChE）催化可卡因水解。具有更高催化活性的抗（-）-可卡因的BChE突变体非常适合用作人体内的外源酶。为了开发高活性突变体的合理设计方法，我们使用扩展的自由能微扰（FEP）程序，对过渡态结构的各种突变以及相应的游离酶结构进行了FEP模拟。对游离酶和过渡态结构突变的FEP模拟使我们能够计算出自由能变化从游离酶（BChE）到过渡态的突变引起的变化，从而从理论上预测催化效率（k(cat)/K(M)）的突变引起的变化。计算预测得到了湿实验获得的动力学数据的支持，表明基于FEP的计算设计方法有望用于合理设计酶的高活性突变体。在本研究中设计和发现的一种BChE突变体与野生型BChE相比，对（-）-可卡因的催化效率提高了约1800倍。这种高活性突变体可能具有治疗价值。
