预反应态模型浅析：催化活性和近过渡态分子模拟

doi:10.12211/2096-8280.2021-013

摘要/Abstract

摘要：

当今生物合成催化元件超进化分子理性设计的瓶颈在于有限的计算资源、研究时间与催化反应复杂势能面接近无穷无尽的计算需求之间的矛盾。然而，两个前所未有的数据集合有望拓新蛋白质工程人工智能化分子设计，其一是高通量定向进化实验带来的巨量高效突变体序列信息，其二是基于结构生物学的高阶量子力学计算所揭示的全原子飞秒精度反应机制。本文从催化基本理论、米氏复合物近进攻构象、催化循环效率控制点的角度浅析预反应态模型的基本概念和应用。预反应态模型尝试利用在低反应势垒生物化学反应中内禀的近进攻构象与过渡态具有相近的物理化学稳定性，弹性地选择与催化元件进化目标相关的关键过渡态，利用经典分子动力学模拟分析近过渡态的活性构象布居数与远端突变、底物结构、实验条件的关系。预反应态分析的基本流程为：首先，基于高阶量子力学反应势能面提取催化中心关键过渡态的结构特征；其次，从高精度蛋白质三维结构出发，结合氨基酸质子化生物信息学预测工具构建出关键过渡态对应的近进攻态活性构象；最后，利用过渡态结构特征设定分子动力学模拟初始约束条件，并逐步取消约束条件测试预反应态随氨基酸突变和底物变化的稳定性变化，以近进攻构象在预反应态轨迹中布居数作为“预反应态-酶活”半定量相关系数，从预反应态稳定性中挖掘酶与底物的适配图谱。当前在预反应态动态结构与酶活的定量关系分析上还有诸多难题亟待突破，利用高通量高阶量子化学再采样计算、结合机器学习人工智能分析代表了预反应态模型的发展方向。

关键词: 预反应态, 近进攻构象, 催化循环, 突变效应, 底物适配性, 分子动力学模拟

Abstract:

The bottleneck of enzyme design for biosynthetic elements lies in the incompetence of the limited computing resources with demanding for an in-depth computation on complicated potential energy surfaces of catalytic reactions. However, two unprecedented achievements are expected to expand artificial intelligence machine learning in protein engineering-one is a variety of high-efficient mutants brought by high-throughput directed evolution experiments, and the other is the high-quality molecular simulation of all-atom with femtosecond precision revealed by ab initio quantum mechanics calculation and three-dimensional structural information. This work briefly describes the basic concept and application of the pre-reaction state (PRS) model from the perspectives of the fundamental enzyme theories, the near-attack conformation of Michealis complex, and the control points of the catalytic cycle efficiency. The pre-reaction state model tries to use the intrinsic features of biochemical reactions with low activation energy in which transition state and pre-reaction states share similar physiochemical stability, flexibly selects the rate-determining transition states related to the evolutional goal of the catalytic element, and employs classical molecular dynamics simulations to understand the relationship of active conformation population with distal mutations, substrate spectrum, and experimental conditions. The general pre-reaction state protocol is: first, the near-transition state structural features are extracted from the high-level quantum-mechanical calculation on the rate-determining transition structures; then the PRS molecular dynamic simulations are collected from the restrained to the free state, which is used to study the adaptability between mutants and substrates. The population in the PRS trajectory is used as a semi-quantitative correlation coefficient of “pre-reaction state-enzyme activity” （PRS-EA）, and the adaptation map of enzyme and substrate is mined from the pre-reaction state stability. Although the mechanism-based pre-reaction state analysis provides an insightful rationale at atom levels as a post-NAC approach, the quantitative relationship between the PRS structure and enzymatic reaction cannot be fully illustrated owing to the ambiguity of the PRS constraint, the repeatability of molecular dynamics simulation, and the arbitrariness of reactive population. The high throughput quantum calculation for transition state samplings and machine learning and artificial intelligence could be integrated to unveil the quantitative structure-activity relationship, paving a way for the practical applications of pre-reaction state in protein engineering.

Key words: pre-reaction state, near attack conformation, catalytic cycle, mutation effect, substrate adaptability, molecular dynamics simulation

中图分类号:

Q816

SIM Byuri, 赵一雷. 预反应态模型浅析：催化活性和近过渡态分子模拟[J]. 合成生物学, 2022, 3(3): 567-586.

Byuri SIM, Yilei ZHAO. Assessment on the pre-reaction state of enzyme: could we understand catalytic activity with near transition-state molecular dynamic simulation?-a review[J]. Synthetic Biology Journal, 2022, 3(3): 567-586.

图/表 5

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研究对象	预反应态所选择的控制点	实例
红霉素DEBS硫酯酶	底物上载后共价复合物水解和环化途径选择	与反应位点间隔4～5个化学键的碳原子对反应中心环化和水解活性结构的远程作用^[95] PRS结构参数：N_H259-O_H距离、O_H-C距离
苦霉素硫酯酶	底物上载后共价复合物水解和环化途径选择	12元和14元大环成环对反应中心环化和水解活性结构的影响^[96] PRS结构参数：N_H268-O_H距离、O_H-C距离
变构霉素硫酯酶/苦霉素硫酯酶	底物上载后共价复合物水解和环化途径选择	变构霉素生物合成时环化释放受阻，利用异源硫酯酶调整环化水解选择性，适配性分析^[97] 变构霉素硫酯酶PRS结构参数：N_H255-O_H距离、O_H-C距离苦霉素硫酯酶PRS结构参数：N_H268-O_H距离、O_H-C距离
匹马霉素硫酯酶	底物上载后共价复合物水解和环化途径选择	与反应位点间隔11～13个化学键的碳原子修饰对反应中心环化和水解活性结构的远程作用^[98] PRS结构参数：N_H261-O_H距离、O_H-C距离
醇脱氢酶KpADH	质子偶合的负氢原子转移关键过渡态	高效突变体与野生型反应中心的活性构象分布变化^[101] PRS结构参数：H_Y164-O距离、H_NADPH-C距离
醇脱氢酶CpRCR	质子偶合的负氢原子转移关键过渡态	高效突变体与野生型反应中心的活性构象分布变化^[103] PRS结构参数：H_S46-O距离、H_NADPH-C距离
DNA甲基化酶3A	SAM甲基转移到DNA-酶共价结合中间体	白血病高发的R882H远端突变对活性中心SAM甲基转移的影响^[106] PRS结构参数：C_SAM-C距离
脱羧酶TyDC	PLP辅因子与底物的共价加合物C－C键断裂	通过H98、H251控制共价加合物的反应前线轨道布局^[108] PRS结构参数：CCαN_SB角度，CCαN_SBC_4’二面角
酰胺水解酶NiHyuC	碳四面体氧负离子关键过渡态	高效突变体对活化后巯基与被进攻的羧基反应前线轨道布局的影响^[110] PRS结构参数：S_C171-C距离、S_C171CO进攻角
普鲁兰多糖酶	取代反应S_N2过渡态（碳氧键断裂）	高效突变体对[O-C-O]取代反应前线轨道布局的影响^[112] PRS结构参数：O_D619-C距离、O_D619CO夹角
MERS和SARS冠状病毒主蛋白水解酶	碳四面体氧负离子关键过渡态	两种病毒主蛋白水解酶的关键反应前线轨道布局^[114] MERS病毒PRS结构参数：S_C148-C距离、S_C148CO进攻角 SARS病毒PRS结构参数：S_C145-C距离、S_C145CO进攻角

研究对象	预反应态所选择的控制点	实例
红霉素DEBS硫酯酶	底物上载后共价复合物水解和环化途径选择	与反应位点间隔4～5个化学键的碳原子对反应中心环化和水解活性结构的远程作用^[95] PRS结构参数：N_H259-O_H距离、O_H-C距离
苦霉素硫酯酶	底物上载后共价复合物水解和环化途径选择	12元和14元大环成环对反应中心环化和水解活性结构的影响^[96] PRS结构参数：N_H268-O_H距离、O_H-C距离
变构霉素硫酯酶/苦霉素硫酯酶	底物上载后共价复合物水解和环化途径选择	变构霉素生物合成时环化释放受阻，利用异源硫酯酶调整环化水解选择性，适配性分析^[97] 变构霉素硫酯酶PRS结构参数：N_H255-O_H距离、O_H-C距离苦霉素硫酯酶PRS结构参数：N_H268-O_H距离、O_H-C距离
匹马霉素硫酯酶	底物上载后共价复合物水解和环化途径选择	与反应位点间隔11～13个化学键的碳原子修饰对反应中心环化和水解活性结构的远程作用^[98] PRS结构参数：N_H261-O_H距离、O_H-C距离
醇脱氢酶KpADH	质子偶合的负氢原子转移关键过渡态	高效突变体与野生型反应中心的活性构象分布变化^[101] PRS结构参数：H_Y164-O距离、H_NADPH-C距离
醇脱氢酶CpRCR	质子偶合的负氢原子转移关键过渡态	高效突变体与野生型反应中心的活性构象分布变化^[103] PRS结构参数：H_S46-O距离、H_NADPH-C距离
DNA甲基化酶3A	SAM甲基转移到DNA-酶共价结合中间体	白血病高发的R882H远端突变对活性中心SAM甲基转移的影响^[106] PRS结构参数：C_SAM-C距离
脱羧酶TyDC	PLP辅因子与底物的共价加合物C－C键断裂	通过H98、H251控制共价加合物的反应前线轨道布局^[108] PRS结构参数：CCαN_SB角度，CCαN_SBC_4’二面角
酰胺水解酶NiHyuC	碳四面体氧负离子关键过渡态	高效突变体对活化后巯基与被进攻的羧基反应前线轨道布局的影响^[110] PRS结构参数：S_C171-C距离、S_C171CO进攻角
普鲁兰多糖酶	取代反应S_N2过渡态（碳氧键断裂）	高效突变体对[O-C-O]取代反应前线轨道布局的影响^[112] PRS结构参数：O_D619-C距离、O_D619CO夹角
MERS和SARS冠状病毒主蛋白水解酶	碳四面体氧负离子关键过渡态	两种病毒主蛋白水解酶的关键反应前线轨道布局^[114] MERS病毒PRS结构参数：S_C148-C距离、S_C148CO进攻角 SARS病毒PRS结构参数：S_C145-C距离、S_C145CO进攻角

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