A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB
A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB
复制标题
DOI：
10.1074/jbc.m112.377853
复制
发表时间：
2012-08-17
影响因子：
4.8
通讯作者：
Wade, Rebecca C.
中科院分区：
文献类型：
--
作者：
Biedermannova, Lada;Prokop, Zbynek;Wade, Rebecca C.
许多酶具有埋藏的活性位点。连接活性中心与本体溶剂的隧道的性质影响配体结合和解结合以及催化性能。在这里，我们调查的配体通道中的卤代烷脱卤酶LinB和取代亮氨酸的效果由一个庞大的色氨酸在隧道衬砌的位置。瞬态动力学实验表明，突变显着减缓产品的释放速率。此外，溴离子的释放机制从野生型酶的一步过程改变为突变体的两步过程。溴离子释放的速率常数对应于整体稳态周转速率常数，表明产品释放成为突变体中催化的限速步骤。我们解释的实验结果，通过调查计算过程的分子细节。从分子动力学模拟与隧道检测软件的轨迹分析揭示了可用于配体出口的隧道的差异。相应的差异被认为是在模拟产品出口使用专门的增强抽样技术。自由能势垒的差异，获得使用潜在的平均力计算溴离子的出口是在良好的协议与从瞬态动力学实验中获得的速率的差异。溴离子与引入的色氨酸的相互作用被证明会影响其通过的自由能垒。这项研究展示了酶催化循环和反应动力学的机制如何通过修改蛋白质隧道来工程化。
Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.