Radhakrishnan Ravi, Schlick Tamar
Department of Bioengineering, University of Pennsylvania, 240 Skirkanich Hall, 210 S. 33rd Street, Philadelphia, PA, USA.
Biochem Biophys Res Commun. 2006 Nov 24;350(3):521-9. doi: 10.1016/j.bbrc.2006.09.059. Epub 2006 Sep 25.
Tracking the structural and energetic changes in the pathways of DNA replication and repair is central to the understanding of these important processes. Here we report favorable mechanisms of the polymerase-catalyzed phosphoryl transfer reactions corresponding to correct and incorrect nucleotide incorporations in the DNA by using a novel protocol involving energy minimizations, dynamics simulations, quasi-harmonic free energy calculations, and mixed quantum mechanics/molecular mechanics dynamics simulations. Though the pathway proposed may not be unique and invites variations, geometric and energetic arguments support the series of transient intermediates in the phosphoryl transfer pathways uncovered here for both the G:C and G:A systems involving a Grotthuss hopping mechanism of proton transfer between water molecules and the three conserved aspartate residues in pol beta's active-site. In the G:C system, the rate-limiting step is the initial proton hop with a free energy of activation of at least 17 kcal/mol, which corresponds closely to measured k(pol) values. Fidelity discrimination in pol beta can be explained by a significant loss of stability of the closed ternary complex of the enzyme in the G:A system and much higher activation energy of the initial step of nucleophilic attack, namely deprotonation of terminal DNA primer O3'H group. Thus, subtle differences in the enzyme active-site between matched and mismatched base pairs generate significant differences in catalytic performance.
追踪DNA复制和修复途径中的结构和能量变化是理解这些重要过程的核心。在这里，我们通过使用一种新的协议，包括能量最小化、动力学模拟、准谐波自由能计算和混合量子力学/分子力学动力学模拟，报告了与DNA中正确和错误核苷酸掺入相对应的聚合酶催化的磷酰基转移反应的有利机制。尽管所提出的途径可能不是唯一的，并且可能存在变化，但几何和能量方面的论据支持了这里发现的磷酰基转移途径中的一系列瞬态中间体，对于G:C和G:A系统，该途径涉及水分子之间质子转移的Grotthuss跳跃机制以及pol β活性位点中的三个保守天冬氨酸残基。在G:C系统中，限速步骤是初始质子跳跃，活化自由能至少为17千卡/摩尔，这与测得的k(pol)值密切对应。pol β中的保真度区分可以通过G:A系统中酶的封闭三元复合物稳定性的显著丧失以及亲核攻击初始步骤（即末端DNA引物O3'H基团的去质子化）的更高活化能来解释。因此，匹配和错配碱基对之间酶活性位点的细微差异会导致催化性能的显著差异。
