Alberts Ian L, Wang Yanli, Schlick Tamar
Department of Chemistry and Courant Institute of Mathematical Sciences, 251 Mercer Street, New York University, New York, New York 10012, USA.
J Am Chem Soc. 2007 Sep 12;129(36):11100-10. doi: 10.1021/ja071533b. Epub 2007 Aug 16.
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(Palpha) of the incoming nucleotide, (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 nucleotide 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 nucleotide specificity differences across polymerase families, in terms of the concept of active site preorganization or the so-called "pre-chemistry avenue".
DNA聚合酶是细胞中负责复制和修复DNA的复杂机制的关键组成部分。在原子水平上识别DNA聚合酶的作用机制对于揭示保真度差异的起源至关重要。哺乳动物DNA聚合酶β（pol β）是X家族中的一个小分子量（39 kDa）成员，是研究聚合酶机制的一个优秀模型系统。在此，我们在一个一致的理论框架内，探索了pol β核苷酸转移反应的几种可行的低能途径，以研究正确（根据沃森-克里克氢键）的G:C碱基配对与错误的G:G配对情况。我们在一个受限能量最小化协议中使用混合量子力学/分子力学（QM/MM）技术，不仅有效地模拟了反应核心，还模拟了酶促环境的其余部分以及明确溶剂对反应的影响。假定的途径包括从末端DNA引物的O3'H基团进行初始质子抽取、亲核攻击以延长DNA引物链以及焦磷酸的消除。特别是，我们分析了初始去质子化步骤的几种可能途径：（i）直接转移到进入核苷酸的磷酸氧O(Pα)上，（ii）直接转移到活性位点的天冬氨酸基团上，以及（iii）转移到明确的水分子上。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分子，随后质子迁移到活性位点的天冬氨酸残基上，最后迁移到离去的焦磷酸基团上，活化能约为15千卡/摩尔。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比步骤（iii）高7千卡/摩尔和11千卡/摩尔。总体而言，正确和错误核苷酸情况的速率决定步骤都是与在磷酸中心的亲核攻击协同进行的初始去质子化；然而，由于活性位点结构扭曲，我们得到的错配G:G情况的活化能比匹配的G:C复合物高5千卡/摩尔。综上所述，我们的结果支持了其他报道的机制，并有助于根据活性位点预组织或所谓的“预化学途径”的概念，定义一个解释不同聚合酶家族核苷酸特异性差异的框架。
