Matute Ricardo A, Yoon Hanwool, Warshel Arieh
Department of Chemistry, University of Southern California, 418 SGM Building, 3620 McClintock Avenue, Los Angeles, California, 90089-1062.
Proteins. 2016 Nov;84(11):1644-1657. doi: 10.1002/prot.25106. Epub 2016 Aug 24.
Elucidating the catalytic mechanism of DNA polymerase is crucial for a progress in the understanding of the control of replication fidelity. This work tries to advance the mechanistic understanding by analyzing the observed effect of mutations of the acidic groups in the active site of Polymerase β as well as the pH effect on the rate constant. The analysis involves both empirical valence bond (EVB) free energy calculations and considerations of the observed pH dependence of the reaction. The combined analysis indicates that the proton transfer (PT) from the nucleophilic O3' has two possible pathways, one to D256 and the second to the bulk. We concluded based on calculations and the experimental pH profile that the most likely path for the wild-type (WT) and the D256E and D256A mutants is a PT to the bulk, although the WT may also use a PT to Asp 256. Our analysis highlights the need for very extensive sampling in the calculations of the activation barrier and also clearly shows that ab initio QM/MM calculations that do not involve extensive sampling are unlikely to give a clear quantitative picture of the reaction mechanism. Proteins 2016; 84:1644-1657. © 2016 Wiley Periodicals, Inc.
阐明DNA聚合酶的催化机制对于深入理解复制保真度的控制至关重要。这项工作试图通过分析聚合酶β活性位点酸性基团突变的观察效应以及pH对速率常数的影响来推进对其机制的理解。分析涉及经验价键（EVB）自由能计算以及对观察到的反应pH依赖性的考量。综合分析表明，亲核O3'的质子转移（PT）有两条可能的途径，一条是转移至D256，另一条是转移至溶剂。基于计算和实验pH曲线，我们得出结论，野生型（WT）以及D256E和D256A突变体最可能的途径是向溶剂的质子转移，尽管野生型也可能利用向天冬氨酸256的质子转移。我们的分析突出了在计算活化能垒时进行非常广泛采样的必要性，并且清楚地表明，不涉及广泛采样的从头算QM/MM计算不太可能给出反应机制的清晰定量图景。《蛋白质》2016年；84:1644 - 1657。© 2016威利期刊公司。
