Editing Domain Motions Preorganize the Synthetic Active Site of Prolyl-tRNA Synthetase.
Editing Domain Motions Preorganize the Synthetic Active Site of Prolyl-tRNA Synthetase.
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DOI：
10.1021/acscatal.0c02381
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发表时间：
2020-09-04
期刊：
影响因子：
12.9
通讯作者：
Bhattacharyya S
中科院分区：
文献类型：
--
作者：
Hu QH;Williams MT;Shulgina I;Fossum CJ;Weeks KM;Adams LM;Reinhardt CR;Musier-Forsyth K;Hati S;Bhattacharyya S
脯氨酰-tRNA 合成酶 (ProRS) 催化脯氨酸与同源 tRNA 共价连接，这是所有生物体中蛋白质合成不可或缺的步骤。 ProRS 是模块化酶，“原核样”ProRS 与“真核样”ProRS 的区别在于，在主催化结构域的基序 2 和基序 3 之间插入了编辑结构域 (INS)。早期的研究表明耦合域动力学的存在可能有助于催化作用。然而，远端、高度移动的 INS 结构域在合成活性位点的催化中所起的作用尚不完全清楚。在本研究中，理论和实验相结合的方法被用来阐明 INS 域动力学的精确作用。进行量子力学/分子力学模拟以模拟粪肠球菌 ProRS 催化 Pro-AMP 的形成。计算了野生型酶形成腺苷酸的能量，并与含有活性位点突变的变体以及缺乏 INS 结构域的缺失突变体进行了对比。综合结果表明，两种不同类型的动力学有助于酶的催化能力。一组运动是 INS 结构域固有的，可导致催化所需的构象预组织。第二种类型的运动源于活性位点残基的静电重组，影响能量分布的高度和宽度，并且在微调基底方向以促进反应碰撞方面具有关键作用。因此，远端域的运动可以预先组织酶的活性位点以优化催化。
Prolyl-tRNA synthetases (ProRSs) catalyze the covalent attachment of proline onto cognate tRNAs, an indispensable step for protein synthesis in all living organisms. ProRSs are modular enzymes and the “prokaryotic-like” ProRSs are distinguished from “eukaryotic-like” ProRSs by the presence of an editing domain (INS) inserted between motifs 2 and 3 of the main catalytic domain. Earlier studies suggested the presence of coupled-domain dynamics could contribute to catalysis; however, the role that the distal, highly mobile INS domain plays in catalysis at the synthetic active site is not completely understood. In the present study, a combination of theoretical and experimental approaches has been used to elucidate the precise role of INS domain dynamics. Quantum mechanical/molecular mechanical simulations were carried out to model catalytic Pro-AMP formation by Enterococcus faecalis ProRS. The energetics of the adenylate formation by the wild-type enzyme was computed and contrasted with variants containing active site mutations, as well as a deletion mutant lacking the INS domain. The combined results revealed that two distinct types of dynamics contribute to the enzyme’s catalytic power. One set of motions is intrinsic to the INS domain and leads to conformational preorganization that is essential for catalysis. A second type of motion, stemming from the electrostatic reorganization of active site residues, impacts the height and width of the energy profile and has a critical role in fine tuning the substrate orientation to facilitate reactive collisions. Thus, motions in a distal domain can preorganize the active site of an enzyme to optimize catalysis.
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