腺苷酸激酶催化循环后期Mg2+转移的分子动力学模拟

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Mg²⁺ + ATP + AMP ⇌ 2ADP + Mg²⁺,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Mg²⁺的参与。最近的实验发现Mg²⁺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。已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Mg²⁺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, Mg²⁺可能只与其中一个ADP分子结合。由于Mg²⁺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Mg²⁺与催化产物ADP分子的配位情况,即Mg²⁺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Mg²⁺)做了分子动力学模拟研究。我们基于metadynamics方法得到了Mg²⁺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, Mg²⁺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, Mg²⁺才会倾向于与NMP结构域的ADP分子结合。另外,我们也刻画了Mg²⁺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。

Metadynamics Simulations of Mg2+Transfer in the Late Stage of the Adenylate Kinase Catalytic Cycle

Adenylate kinase is a kind of important enzymes which can catalyze the reversible reaction Mg²⁺ + ATP + AMP ⇌ 2ADP + Mg²⁺where the Mg²⁺ coordination around the active site plays a crucial role. It was shown experimentally that one Mg²⁺ ion can coordinate to both ADP molecules right after the chemical step of the catalytic reaction. During the substrate releasing and separation, the Mg²⁺ may transfer to one of the ADP molecules. However, it is unclear which ADP molecule binds with the Mg²⁺ during the substrate releasing. In this work, by using metadynamics method, we conducted molecular simulations on the adenylate kinase complexed with two ADP molecules and one Mg²⁺, which corresponds to the postcatalysis enzyme-substrate complex. We constructed the free energy landscapes characterizing the Mg²⁺ transfer to the individual ADP molecules. Our results show that the Mg²⁺ has preference to attach with the ADP molecule of the LID domain. We found that only when the LID domain ADP is protonated, and simultaneously the NMP domain ADP is deprotonated, the Mg²⁺ tends to attach with the NMP domain ADP. We also characterized the ligand exchange and dehydration processes during the Mg²⁺ transfer. Our results provide insights into the molecular process during the late state of the adenylate kinase catalytic cycle.