Henrique F. Carvalho, Luuk Mestrom, Ulf Hanefeld and Jürgen Pleiss*,
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引用次数: 0
摘要
烟曲霉分枝杆菌中的酰基转移酶是一种多功能酶,它在水介质中催化酯类的酯化反应,这是因为在动力学上合成反应比热力学上的水解反应更优先。在活性八聚体中,活性位点被深埋,并通过长而疏水的底物通道与蛋白质表面相连。分子动力学模拟和马尔可夫状态模型研究了通道在控制催化活性和底物特异性方面的作用,并比较了酰基供体、受体和水结合的热力学和动力学。尽管底物通道具有疏水性,但通道中仍存在水,并与酰基受体竞争进入活性位点。在 10 至 500 mM 的浓度范围内,分析了进入通道的结合自由能曲线以及丁醇、苯甲醇和乙酸乙烯酯的通量,并与水进行了比较。通量在酒精浓度为 50-100 mM 时达到最大值,这与实验观察结果一致。在最大值时,酒精的通量接近水通量的 50%,这就解释了为什么酯交换率比水解率高。造成这种效应的分子原因是酒精分子沿着通路聚集。利用马尔可夫状态模型进行的大量分子动力学模拟和轨迹分析,使人们深入了解了通道通过控制竞争底物的进入和结合在活性和特异性方面的作用。
Beyond the Chemical Step: The Role of Substrate Access in Acyltransferase from Mycobacterium smegmatis
Acyltransferase from Mycobacterium smegmatis is a versatile enzyme, which catalyzes the transesterification of esters in aqueous media due to a kinetic preference of the synthesis reaction over the thermodynamically favored hydrolysis reaction. In the active octamer, the active site is deeply buried and connected to the protein surface by long and hydrophobic substrate access channels. The role of the access channel in controlling catalytic activity and substrate specificity was investigated by molecular dynamics simulations and Markov-state models, and the thermodynamics and kinetics of binding of acyl donors, acceptors, and water were compared. Despite the hydrophobic nature of the substrate access channel, water is present in the channel and competes with the acyl acceptors for access to the active site. The binding free energy profiles in the access channel and the flux of butyl and benzyl alcohol and vinyl acetate were analyzed in the concentration range between 10 and 500 mM and compared to water. The flux showed a maximum at an alcohol concentration of 50–100 mM, in agreement with experimental observations. At the maximum, the flux of alcohol approaches 50% of the flux of water, which explains the high transesterification rate as compared to hydrolysis. The molecular origin of this effect is due to the accumulation of alcohol molecules along the access channel. Extensive molecular dynamics simulations and analysis of trajectories by a Markov-state model provided insights into the role of the access channel in activity and specificity by controlling access and binding of competing substrates.
期刊介绍:
ACS Catalysis is an esteemed journal that publishes original research in the fields of heterogeneous catalysis, molecular catalysis, and biocatalysis. It offers broad coverage across diverse areas such as life sciences, organometallics and synthesis, photochemistry and electrochemistry, drug discovery and synthesis, materials science, environmental protection, polymer discovery and synthesis, and energy and fuels.
The scope of the journal is to showcase innovative work in various aspects of catalysis. This includes new reactions and novel synthetic approaches utilizing known catalysts, the discovery or modification of new catalysts, elucidation of catalytic mechanisms through cutting-edge investigations, practical enhancements of existing processes, as well as conceptual advances in the field. Contributions to ACS Catalysis can encompass both experimental and theoretical research focused on catalytic molecules, macromolecules, and materials that exhibit catalytic turnover.