The transition state for peptide bond formation reveals the ribosome as a water trap
The transition state for peptide bond formation reveals the ribosome as a water trap
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DOI：
10.1073/pnas.0914192107
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发表时间：
2010-02-02
影响因子：
11.1
通讯作者：
Aqvist, Johan
中科院分区：
文献类型：
--
作者：
Wallin, Goran;Aqvist, Johan
在阐明核糖体上的肽键形成过程的最新进展导致了质子穿梭机制的概念，其中P-位tRNA的2‘-羟基在调节亲核基团和离开基团之间的质子转移中起关键作用，而核糖体基团并不积极参与反应。尽管取得了这些进展，但肽转移过渡态的详细性质以及几个捕获的水分子在肽转移酶中心的作用仍然是主要的悬而未决的问题。在这里，我们使用高级量子化学从头计算来定位和表征反应的全局过渡态，由包含反应中心所有关键元素的分子模型描述。计算的活化热和结构与实验数据很好地吻合，并指出了一个八元“双质子穿梭”机制的可行性，在计算机模拟和晶体结构中都观察到了辅助水分子的积极参与。第二个保守的水分子对稳定底物氧离子上的显影负电荷起着关键作用，它的存在在活化热和活化熵方面都是有利的。对于六元和八元机制计算的过渡态总是晚的，并且不涉及攻击氨基上的显着电荷发展。与这张图一致的预测的动力学同位素效应类似于在溶液中观察到的非催化酯氨解反应。
Recent progress in elucidating the peptide bond formation process on the ribosome has led to notion of a proton shuttle mechanism where the 2'-hydroxyl group of the P-site tRNA plays a key role in mediating proton transfer between the nucleophile and leaving group, whereas ribosomal groups do not actively participate in the reaction. Despite these advances, the detailed nature of the transition state for peptidyl transfer and the role of several trapped water molecules in the peptidyl transferase center remain major open questions. Here, we employ high-level quantum chemical ab initio calculations to locate and characterize global transition states for the reaction, described by a molecular model encompassing all the key elements of the reaction center. The calculated activation enthalpy as well as structures are in excellent agreement with experimental data and point to feasibility of an eight-membered "double proton shuttle" mechanism in which an auxiliary water molecule, observed both in computer simulations and crystal structures, actively participates. A second conserved water molecule is found to be of key importance for stabilizing developing negative charge on the substrate oxyanion and its presence is catalytically favorable both in terms of activation enthalpy and entropy. Transition states calculated both for six-and eight-membered mechanisms are invariably late and do not involve significant charge development on the attacking amino group. Predicted kinetic isotope effects consistent with this picture are similar to those observed for uncatalyzed ester aminolysis reactions in solution.