Dutta Saheb, Chandra Amalendu
Department of Chemistry, Institute of Technology Kanpur, Kanpur, Uttar Pradesh 208016, India.
J Chem Inf Model. 2023 Mar 27;63(6):1819-1832. doi: 10.1021/acs.jcim.2c01332. Epub 2023 Mar 9.
Aspartyl-tRNA synthetase catalyzes the attachment of aspartic acid to its cognate tRNA by the aminoacylation reaction during the initiation of the protein biosynthesis process. In the second step of the aminoacylation reaction, known as the charging step, the aspartate moiety is transferred from aspartyl-adenylate to the 3'-OH of A76 of tRNA through a proton transfer process. We have investigated different pathways for the charging step through three separate QM/MM simulations combined with the enhanced sampling method of well-sliced metadynamics and found out the most feasible pathway for the reaction at the active site of the enzyme. In the charging reaction, both the phosphate group and the ammonium group after deprotonation can potentially act as a base for proton transfer in the substrate-assisted mechanism. We have considered three possible mechanisms involving different pathways of proton transfer, and only one of them is determined to be enzymatically feasible. The free energy landscape along reaction coordinates where the phosphate group acts as the general base showed that, in the absence of water, the barrier height is 52.6 kcal/mol. The free energy barrier is reduced to 39.7 kcal/mol when the active site water molecules are also treated quantum mechanically, thus allowing a water mediated proton transfer. The charging reaction involving the ammonium group of the aspartyl adenylate is found to follow a path where first a proton from the ammonium group moves to a water in the vicinity forming a hydronium ion (HO) and NH group. The hydronium ion subsequently passes the proton to the Asp233 residue, thus minimizing the chance of back proton transfer from hydronium to the NH group. The neutral NH group subsequently takes the proton from the O3' of A76 with a free energy barrier of 10.7 kcal/mol. In the next step, the deprotonated O3' makes a nucleophilic attack to the carbonyl carbon forming a tetrahedral transition state with a free energy barrier of 24.8 kcal/mol. Thus, the present work shows that the charging step proceeds through a multiple proton transfer mechanism where the amino group formed after deprotonation acts as the base to capture a proton from O3' of A76 rather than the phosphate group. The current study also shows the important role played by Asp233 in the proton transfer process.
天冬氨酰 - tRNA合成酶在蛋白质生物合成过程起始阶段，通过氨酰化反应催化天冬氨酸连接到其对应的tRNA上。在氨酰化反应的第二步，即负载步骤中，天冬氨酸部分通过质子转移过程从天冬氨酰 - 腺苷酸转移到tRNA的A76的3'-OH上。我们通过三个独立的QM/MM模拟结合精细切片元动力学增强采样方法，研究了负载步骤的不同途径，并找出了该酶活性位点上反应最可行的途径。在负载反应中，去质子化后的磷酸基团和铵基团都有可能在底物辅助机制中作为质子转移的碱。我们考虑了三种涉及不同质子转移途径的可能机制，其中只有一种被确定在酶促反应中是可行的。沿着以磷酸基团作为通用碱的反应坐标的自由能景观显示，在无水情况下，势垒高度为52.6千卡/摩尔。当活性位点水分子也进行量子力学处理时，自由能垒降低到39.7千卡/摩尔，从而允许水介导的质子转移。发现涉及天冬氨酰腺苷酸铵基团的负载反应遵循这样一条路径：首先铵基团的一个质子转移到附近的一个水分子上，形成水合氢离子（H₃O⁺）和NH基团。随后水合氢离子将质子传递给Asp233残基，从而使水合氢离子将质子反向转移到NH基团的可能性最小化。中性的NH基团随后从A76的O3'获取质子，自由能垒为10.7千卡/摩尔。在下一步中，去质子化的O3'对羰基碳进行亲核攻击，形成一个自由能垒为24.8千卡/摩尔的四面体过渡态。因此，目前的工作表明负载步骤通过多质子转移机制进行，其中去质子化后形成的氨基作为碱从A76的O3'捕获质子，而不是磷酸基团。当前的研究还显示了Asp233在质子转移过程中所起的重要作用。
