Department of Chemistry, Indian Institute of Technology Kanpur, Uttar Pradesh, India 208016.
J Phys Chem B. 2022 Aug 11;126(31):5821-5831. doi: 10.1021/acs.jpcb.2c03843. Epub 2022 Jul 27.
The process of protein biosynthesis is initiated by the aminoacylation process where a transfer ribonucleic acid (tRNA) is charged by the attachment of its cognate amino acid at the active site of the corresponding aminoacyl tRNA synthetase enzyme. The first step of the aminoacylation process, known as the adenylation reaction, involves activation of the cognate amino acid where it reacts with a molecule of adenosine triphosphate (ATP) at the active site of the enzyme to form the aminoacyl adenylate and inorganic pyrophosphate. In the current work, we have investigated the adenylation reaction between aspartic acid and ATP at the active site of the fully solvated aspartyl tRNA synthetase (AspRS) from in aqueous medium at room temperature through hybrid quantum mechanical/molecular mechanical (QM/MM) simulations combined with enhanced sampling methods of well-tempered and well-sliced metadynamics. The objective of the present work is to study the associated free energy landscape and reaction barrier and also to explore the effects of active site mutation on the free energy surface of the reaction. The current calculations include finite temperature effects on free energy profiles. In particular, apart from contributions of interaction energies, the current calculations also include contributions of conformational, vibrational, and translational entropy of active site residues, substrates, and also the rest of the solvated protein and surrounding water into the free energy calculations. The present QM/MM metadynamics simulations predict a free energy barrier of 23.35 and 23.5 kcal/mol for two different metadynamics methods used to perform the reaction at the active site of the wild type enzyme. The free energy barrier increases to 30.6 kcal/mol when Arg217, which is an important conserved residue of the wild type enzyme at its active site, is mutated by alanine. These free energy results including the effect of mutation compare reasonably well with those of kinetic experiments that are available in the literature. The current work also provides molecular details of structural changes of the reactants and surroundings as the system dynamically evolves along the reaction pathway from reactant to the product state through QM/MM metadynamics simulations.
蛋白质生物合成的过程是由氨酰基转移 RNA(tRNA)的氨酰化过程启动的，其中 tRNA 在相应的氨酰基 tRNA 合成酶酶的活性部位与其同源氨基酸连接。氨酰化过程的第一步，即腺苷酸化反应，涉及到同源氨基酸的活化，其中它在酶的活性部位与三磷酸腺苷(ATP)分子反应，形成氨酰腺苷酸和无机焦磷酸。在目前的工作中，我们通过混合量子力学/分子力学(QM/MM)模拟结合增强采样方法，如 well-tempered 和 well-sliced metadynamics，研究了在室温下在完全水合的天冬氨酰 tRNA 合成酶(AspRS)的活性部位中天冬氨酸与 ATP 之间的腺苷酸化反应。本工作的目的是研究相关的自由能景观和反应势垒，并探索活性部位突变对反应自由能表面的影响。目前的计算包括对自由能曲线的有限温度效应。特别是，除了相互作用能的贡献外，目前的计算还包括活性部位残基、底物以及周围水溶剂化的蛋白质和水的构象、振动和平移熵的贡献到自由能计算中。目前的 QM/MM metadynamics 模拟预测，在野生型酶的活性部位使用两种不同的 metadynamics 方法进行反应时，自由能势垒分别为 23.35 和 23.5 kcal/mol。当 Arg217（其是野生型酶活性部位的一个重要保守残基）突变为丙氨酸时，自由能势垒增加到 30.6 kcal/mol。这些自由能结果包括突变的影响与文献中可用的动力学实验结果相当吻合。目前的工作还通过 QM/MM metadynamics 模拟提供了反应物和周围环境结构变化的分子细节，因为系统沿着反应途径从反应物动态演化到产物状态。
