Dissecting Conformational Dynamics-Modulated Enzyme Catalysis with Single-Molecule FRET
Dissecting Conformational Dynamics-Modulated Enzyme Catalysis with Single-Molecule FRET
复制标题
用单分子 FRET 剖析构象动力学调节酶催化
DOI：
10.1021/acs.jpcb.8b02374
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Wenning Wang
中科院分区：
文献类型：
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作者：
Shaowen Wu;Jianwei Liu;Wenning Wang
酶蛋白的构象变化常常伴随着催化反应并调节酶的活性。单分子技术是研究这些复杂情况下酶催化机理的有力工具。然而，在单分子检测实验中不能同时监测化学反应循环和构象变化，导致一些关键动力学参数未得到解决。在这里，我们描述了一种方法来提取所有的动力学参数，从全面的单分子FRET（smFRET）测量和模型分析。在smFRET的基础上，我们通过求解动力学模型的速率方程，以smFRET测量的构象状态种群和状态转换速率常数为输入，计算了不可检测的参数。以MalK2 ATP酶为例，说明该方法可以定量地揭示酶催化反应的机理及其耦合的构象动力学。本研究所采用的策略可广泛应用于研究其他酶的构象波动偶联催化。
Conformational changes of enzyme proteins are often coupled with a catalytic reaction and modulate the enzyme activity. Single-molecule technology is a powerful tool to study the mechanism of enzyme catalysis in these complicated cases. However, the chemical reaction cycles and conformational changes could not be monitored simultaneously in a single-molecule detection experiment, resulting in some unresolved key kinetic parameters. Here, we describe a method to extract all of the kinetic parameters from comprehensive single-molecule FRET (smFRET) measurements and model analysis. On the basis of the smFRET, we calculated the undetectable parameters by solving the rate equations of the kinetic model with the input of the smFRET-measured conformational state populations and state-transition rate constants. A case study of MalK2ATPase demonstrates that this method could reveal the quantitative mechanism of the catalytic reaction of the enzyme as well as its coupled conformational dynamics. The strategy employed in this study could be widely applied to investigate the conformational fluctuation-coupled catalysis of other enzymes.