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腺苷酸激酶是一个包含三个结构域(LID结构域、NMP结构域和CORE结构域)的蛋白质分子,其主要作用是催化化学反应Mg~(2+)+ATP+AMP(?)2ADP+Mg~(2+)进而将细胞内ATP分子的浓度维持在合适的范围内。在腺苷酸激酶催化上述化学反应的过程中,需要有Mg~(2+)的参与。最近的实验发现Mg~(2+)不仅参与上述反应的化学步骤,而且对化学反应发生后底物的释放过程至关重要。己有晶体结构数据显示,在催化循环过程的化学反应步骤完成后,一个Mg~(2+)可同时和分别位于LID结构域及NMP结构域的两个ADP分子配位。然而,在底物的释放与分离过程中,Mg~(2+)可能只与其中一个ADP分子结合。由于Mg~(2+)与ADP分子的结合情况会在很大程度上影响作为催化循环限速步骤的底物释放过程,因此人们有必要研究清楚在底物释放前Mg~(2+)与催化产物ADP分子的配位情况,即Mg~(2+)更倾向于与LID结构域的ADP分子结合还是与NMP结构域的ADP分子结合。本文中,我们对催化反应后底物释放前的酶-底物复合物(包含酶、两个ADP分子以及Mg~(2+))做了分子动力学模拟研究。我们基于metadynamics方法得到了Mg~(2+)在两个ADP分子间转移的自由能面,发现在底物分离与释放过程中,Mg~(2+)更倾向于与LID结构域的ADP分子结合。只有当LID结构域的ADP分子被质子化,同时NMP结构域的ADP分子处于去质子化状态时,Mg~(2+)才会倾向于与NMP结构域的ADP分子结合。另外,我们也刻画了Mg~(2+)转移过程中配体交换与脱水过程。本工作的研究结果有助于理解腺苷酸激酶催化循环后期的分子过程。
Adenylate kinase is a protein molecule containing three domains (LID domain, NMP domain and CORE domain). Its main function is to catalyze the chemical reaction of Mg 2+ + ATP + AMP + 2ADP + Mg 2+ in turn maintains the concentration of intracellular ATP molecules within a suitable range. Adenylate kinase in the chemical reaction of the above process, the need for Mg ~ (2+) participation. Recent experiments found that Mg 2+ not only participates in the chemical reaction of the above reaction, but also is crucial for the release of the substrate after the chemical reaction takes place. The crystallographic data have shown that after the chemical reaction step of the catalytic cycle is completed, one Mg 2+ can simultaneously coordinate with two ADP molecules located in the LID domain and the NMP domain, respectively. However, during the process of substrate release and separation, Mg 2+ may bind to only one ADP molecule. Since the binding of Mg 2+ to ADP molecules will largely affect the substrate release process as a rate-limiting step in the catalytic cycle, it is necessary to study clearly the effect of Mg 2+ and The coordination of the catalytic product ADP molecules, that is, Mg 2+ is more likely to bind to ADP molecules in the LID domain or ADP molecules in the NMP domain. In this paper, we performed a molecular dynamics simulation of the enzyme-substrate complex (including the enzyme, two ADP molecules and Mg 2+) before the substrate was released after the catalytic reaction. Based on the metadynamics method, we obtained the free energy surface of Mg 2+ transfer between two ADP molecules and found that Mg 2+ is more likely to bind to ADP molecules in the LID domain during substrate dissociation and release . Only when the ADP molecules of the LID domain are protonated and the ADP molecules of the NMP domain are in a deprotonated state will Mg 2+ tend to bind to the NMP domain ADP molecule. In addition, we also characterize the process of ligand exchange and dehydration in Mg ~ (2+) transfer. The findings of this work contribute to the understanding of the molecular processes that catalyze the later stages of adenylate kinase.