Kinetic Isotope Effects for RNA Cleavage by 2′-O-Transphosphorylation: Nucleophilic Activation by Specific Base
Kinetic Isotope Effects for RNA Cleavage by 2′-O-Transphosphorylation: Nucleophilic Activation by Specific Base
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DOI：
10.1021/ja103550e
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发表时间：
2010-08-25
影响因子：
15
通讯作者：
Anderson, Vernon E.
中科院分区：
文献类型：
--
作者：
Harris, Michael E.;Dai, Qing;Anderson, Vernon E.
为了更好地了解 RNA 链切割过程中催化剂和过渡态之间的相互作用，测量了初级 O-18 动力学同位素效应 (KIE) 和溶剂 D2O 同位素效应，以探讨 RNA 二核苷酸 5'-UpG-3' 碱基催化 2'-O-转磷酸化的机制。在 pH 14 下观察到的亲核 2'-O 和 5'-O 离去基团的 O-18 KIE 相对于具有良好离去基团的磷酸二酯的反应来说都较大，这表明氢氧化物催化的反应具有过渡态 (TS)，其中磷-氧键向离去基团进行高级裂变 (K-18(LG) = 1.034 +/- 0.004），并且 磷-亲核键形成 (K-18(Nuc) = 0.984 +/- 0.004)。 2'-O-转磷酸化速率的 pH 依赖性到 pH 13 以上的 pH 独立相的断点归因于 2'-OH 亲核试剂的 pK(a)。在 pH 12 时观察到较小的亲核试剂 KIE ((18)k(Nuc) = 0.995 +/- 0.004)，这被解释为平衡同位素效应 (约 1.02) 对 2'-羟基亲核试剂去质子化和亲核加成步骤中的内在 KIE (约 0.981) 的综合影响。鉴于在 pH 值与速率曲线的断点以上缺乏溶剂氘同位素效应，氢氧根离子充当一般碱的替代机制被认为不太可能。这些结果代表了对 RNA 链切割过渡态的首次直接分析。主要的 O-18 KIE 结果和缺乏动力溶剂氘同位素效应共同为后期过渡态和特定碱催化的 2'-O 亲核试剂活化提供了强有力的证据。
To better understand the interactions between catalysts and transition states during RNA strand cleavage, primary O-18 kinetic isotope effects (KIEs) and solvent D2O isotope effects were measured to probe the mechanism of base-catalyzed 2'-O-transphosphorylation of the RNA dinucleotide 5'-UpG-3'. The observed O-18 KIEs for the nucleophilic 2'-O and in the 5'-O leaving group at pH 14 are both large relative to reactions of phosphodiesters with good leaving groups, indicating that the reaction catalyzed by hydroxide has a transition state (TS) with advanced phosphorus-oxygen bond fission to the leaving group (K-18(LG) = 1.034 +/- 0.004) and phosphorus-nucleophile bond formation (K-18(Nuc) = 0.984 +/- 0.004). A breakpoint in the pH dependence of the 2'-O-transphosphorylation rate to a pH independent phase above pH 13 has been attributed to the pK(a) of the 2'-OH nucleophile. A smaller nucleophile KIE is observed at pH 12 ((18)k(Nuc) = 0.995 +/- 0.004) that is interpreted as the combined effect of the equilibrium isotope effect (ca. 1.02) on deprotonation of the 2'-hydroxyl nucleophile and the intrinsic KIE on the nucleophilic addition step (ca. 0.981). An alternative mechanism in which the hydroxide ion acts as a general base is considered unlikely given the lack of a solvent deuterium isotope effect above the breakpoint in the pH versus rate profile. These results represent the first direct analysis of the transition state for RNA strand cleavage. The primary O-18 KIE results and the lack of a kinetic solvent deuterium isotope effect together provide strong evidence for a late transition state and 2'-O nucleophile activation by specific base catalysis.