Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polyrnerases
Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polyrnerases
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DOI：
10.1073/pnas.0608952104
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发表时间：
2007-03-13
影响因子：
11.1
通讯作者：
Cameron, Craig E.
中科院分区：
文献类型：
--
作者：
Castro, Christian;Smidansky, Eric;Cameron, Craig E.
大多数核酸聚合酶在前稳定状态下核苷酸结合的限速步骤被认为是构象变化。因此，关于活性位点残基在核苷酸转移化学中的作用的信息很少。对于脊髓灰质炎病毒依赖RNA的RNA聚合酶（3D(pol)），化学反应是部分（Mg2+）或完全（Mn2+）限速的。在这里，我们表明核苷酸转移取决于两个可电离基团，其pK(a)值为7.0或8.2和10.5，这取决于反应中使用的二价阳离子。在预稳定状态下，对核苷酸掺入速率常数存在3 ~ 7的溶剂氘同位素效应；在稳定状态下没有观察到。质子库存实验表明，在反应的限速过渡态中有两个质子被转移，这表明3'-羟基亲核试剂的去质子化和焦磷酸离去基的质子化都发生在形成磷酸二酯键的过渡态中。重要的是，在RB69 DNA依赖的DNA聚合酶、T7 DNA依赖的RNA聚合酶和HIV逆转录酶催化的核苷酸转移反应中，两个质子转移发生在过渡态。在已知聚合酶结构的背景下对这些数据的解释表明，尽管不能最终排除水分子的使用，但3'-OH亲核试剂的去质子化存在一般碱，并且在所有核酸聚合酶中焦磷酸离去基的质子化存在一般酸。这些数据暗示了类似于关联的过渡状态结构。
The rate-limiting step for nucleotide incorporation in the presteady state for most nucleic acid polymerases is thought to be a conformational change. As a result, very little information is available on the role of active-site residues in the chemistry of nucleotidyl transfer. For the poliovirus RNA-dependent RNA polymerase (3D(pol)), chemistry is partially (Mg2+) or completely (Mn2+) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pK(a) values of 7.0 or 8.2 and 10.5, depending upon the divalent cation used in the reaction. A solvent deuterium isotope effect of three to seven was observed on the rate constant for nucleotide incorporation in the pre-steady state; none was observed in the steady state. Proton-inventory experiments were consistent with two protons being transferred during the rate-limiting transition state of the reaction, suggesting that both deprotonation of the 3'-hydroxyl nucleophile and protonation of the pyrophosphate leaving group occur in the transition state for phosphodiester bond formation. Importantly, two proton transfers occur in the transition state for nucleoticlyl-transfer reactions catalyzed by RB69 DNA-dependent DNA polymerase, T7 DNA-dependent RNA polymerase and HIV reverse transcriptase. Interpretation of these data in the context of known polymerase structures suggests the existence of a general base for deprotonation of the 3'-OH nucleophile, although use of a water molecule cannot be ruled out conclusively, and a general acid for protonation of the pyrophosphate leaving group in all nucleic acid polymerases. These data imply an associative-like transition-state structure.