DNA polymerase β catalysis:: Are different mechanisms possible?
DNA polymerase β catalysis:: Are different mechanisms possible?
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DOI：
10.1021/ja071533b
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发表时间：
2007-09-12
影响因子：
15
通讯作者：
Schlick, Tamar
中科院分区：
文献类型：
--
作者：
Alberts, Ian L.;Wang, Yanli;Schlick, Tamar
DNA聚合酶是用于复制和修复DNA的复杂细胞机制的关键组成部分。在原子水平上辨别DNA聚合酶的机制途径对于揭示保真度区分的起源非常重要。哺乳动物DNA聚合酶β（pol β）是X家族的一个小成员（39 kDa），是研究聚合酶机制的一个极好的模型系统。在此，我们在一个一致的理论框架内，针对正确的（根据沃森 - 克里克氢键）G:C碱基配对以及错误的G:G情况，探索了pol β的核苷酸转移反应的几种可行的低能途径。我们在约束能量最小化方案中使用混合量子力学/分子力学（QM/MM）技术，不仅有效地模拟反应核心，还模拟酶环境的其余部分以及显式溶剂对反应的影响。假定的途径包括从末端DNA引物的O3'H基团初始抽取质子、使DNA引物链延伸的亲核攻击以及焦磷酸的消除。特别是，我们分析了初始去质子化步骤的几种可能途径：（i）直接转移到进入的核苷酸的磷酸氧O(P - α)，（ii）直接转移到活性位点的天冬氨酸（Asp）基团，以及（iii）转移到显式水分子。我们发现最可能的初始步骤对应于步骤（iii），即初始去质子化到水，随后质子迁移到活性位点的天冬氨酸残基，最后到离去的焦磷酸基团，活化能约为15 kcal/mol。我们认为初始去质子化步骤（i）和（ii）可能性较小，因为它们的能量分别至少高出7和11 kcal/mol。总体而言，对于正确和错误的核苷酸情况，速率决定步骤都是与在磷酸中心的亲核攻击协同的初始去质子化；然而，由于活性位点结构扭曲，我们得到的错配的G:G情况的活化能比匹配的G:C复合物高5 kcal/mol。综上所述，我们的结果支持其他已报道的机制，并有助于根据活性位点预组织的概念或所谓的“预化学途径”，为解释聚合酶家族之间的核苷酸特异性差异定义一个框架。
DNA polymerases are crucial constituents of the complex cellular machinery for replicating and repairing DNA. Discerning mechanistic pathways of DNA polymerase on the atomic level is important for revealing the origin of fidelity discrimination. Mammalian DNA polymerase beta (pol beta), a small (39 kDa) member of the X-family, represents an excellent model system to investigate polymerase mechanisms. Here, we explore several feasible low-energy pathways of the nucleotide transfer reaction of pol beta for correct (according to Watson-Crick hydrogen bonding) G:C basepairing versus the incorrect G:G case within a consistent theoretical framework. We use mixed quantum mechanics/molecular mechanics (QM/MM) techniques in a constrained energy minimization protocol to effectively model not only the reactive core but also the influence of the rest of the enzymatic environment and explicit solvent on the reaction. The postulated pathways involve initial proton abstraction from the terminal DNA primer O3'H group, nucleophilic attack that extends the DNA primer chain, and elimination of pyrophosphate. In particular, we analyze several possible routes for the initial deprotonation step: (i) direct transfer to a phosphate oxygen O(P-alpha) of the incoming nucleoticle, (ii) direct transfer to an active site Asp group, and (iii) transfer to explicit water molecules. We find that the most probable initial step corresponds to step (iii), involving initial deprotonation to water, which is followed by proton migration to active site Asp residues, and finally to the leaving pyrophosphate group, with an activation energy of about 15 kcal/mol. We argue that initial deprotonation steps (i) and (ii) are less likely as they are at least 7 and 11 kcal/mol, respectively, higher in energy. Overall, the rate-determining step for both the correct and the incorrect nucleoticle cases is the initial deprotonation in concert with nucleophilic attack at the phosphate center; however, the activation energy we obtain for the mismatched G:G case is 5 kcal/mol higher than that of the matched G:C complex, due to active site structural distortions. Taken together, our results support other reported mechanisms and help define a framework for interpreting nucleoticle specificity differences across polymerase families, in terms of the concept of active site preorganization or the so-called "pre-chemistry avenue".