Archaeal threonyl-tRNA synthetase (ThrRS) possesses an editing active site wherein tRNA^Thr that has been misaminoacylated with serine (i.e., Ser-tRNA^Thr) is hydrolytically cleaved to serine and tRNA^Thr. It has been suggested that the free ribose sugar hydroxyl of Ado76 of the tRNA^Thr (_Ado762′OH) is the mechanistic base, promoting hydrolysis by orienting a nucleophilic water near the scissile Ser-tRNA^Thr ester bond. We have performed a computational study, involving molecular dynamics (MD) and hybrid ONIOM quantum mechanics/molecular mechanics (QM/MM) methods, considering all possible editing mechanisms to gain an understanding of the role played by _Ado762′OH group. More specifically, a range of concerted or stepwise mechanisms involving four-, six-, or eight-membered transition structures (total of seven mechanisms) were considered. In addition, these seven mechanisms were fully optimized using three different DFT functionals, namely, B3LYP, M06-2X, and M06-HF. The M06-HF functional gave the most feasible energy barriers followed by the M06-2X functional. The most favorable mechanism proceeds stepwise through two six-membered ring transition states in which the _Ado762′OH group participates, overall, as a shuttle for the proton transfer from the nucleophilic H₂O to the bridging oxygen (_Ado763′O) of the substrate. More specifically, in the first step, which has a barrier of 25.9 kcal/mol, the _Ado762′-OH group accepts a proton from the attacking nucleophilic water while concomitantly transferring its proton onto the substrates C–O_carb center. Then, in the second step, which also proceeds with a barrier of 25.9 kcal/mol, the _Ado762′-OH group transfers its proton on the adjacent _Ado763′-oxygen, cleaving the scissile C_carb–O3′_Ado76 bond, while concomitantly accepting a proton from the previously formed C–O_carbH group.

中文翻译：

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_阐明Ado76 2'OH在古细菌苏氨酸-tRNA合成酶的转移后编辑中所起的关键作用

古细菌苏氨酰tRNA合成酶酶（ThrRS）具有一个编辑的活性位点，其中的tRNA^苏氨酸已被misaminoacylated用丝氨酸（即，丝氨酸酰-tRNA^苏氨酸）被水解裂解以丝氨酸和tRNA^苏氨酸。已经提出，tRNA ^Thr的Ado76的游离核糖糖羟基（_Ado76 2'OH）是机制碱基，通过使易裂的Ser-tRNA ^Thr酯键附近的亲核水取向来促进水解。我们进行了一项计算研究，涉及分子动力学（MD）和混合ONIOM量子力学/分子力学（QM / MM）方法，并考虑了所有可能的编辑机制以了解_Ado76的作用2′OH基团。更具体地说，考虑了涉及四元，六元或八元过渡结构（总共七个机制）的一系列协调或逐步机制。此外，使用三种不同的DFT功能（即B3LYP，M06-2X和M06-HF）对这七个机制进行了完全优化。M06-HF功能提供了最可行的能垒，其次是M06-2X功能。最有利的机制逐步进行，通过两个六元环跃迁状态，其中_Ado76 2'OH基团总体上作为质子从亲核H ₂ O转移至桥联氧（_Ado76 3'O）的_穿梭体而参与。基板。更具体地，在具有25.9 kcal / mol的势垒的第一步中，_Ado76 2'-OH基团从_侵蚀性亲核水中接受质子，同时将其质子转移到基质C–O_碳中心上。然后，在第二步骤中，这也与25.9阻挡前进千卡/摩尔，所述_Ado76 2'-OH基团转移其上的相邻质子_Ado76 3'-氧，切割易切断Ç_{碳水化合物}-O3' _Ado76键，而同时接受先前形成的C–O _carb H基团的质子。