Klvaňa Martin, Bren Urban, Florián Jan
Laboratory of Physical Chemistry and Chemical Thermodynamics, Faculty of Chemistry and Chemical Technology, University of Maribor , Smetanova ulica 17, 2000 Maribor, Slovenia.
Department of Chemistry and Biochemistry, Loyola University Chicago , 1032 W. Sheridan Road, Chicago, Illinois 60660, United States.
J Phys Chem B. 2016 Dec 29;120(51):13017-13030. doi: 10.1021/acs.jpcb.6b08581. Epub 2016 Dec 19.
Human X-family DNA polymerases β (Polβ) and λ (Polλ) catalyze the nucleotidyl-transfer reaction in the base excision repair pathway of the cellular DNA damage response. Using empirical valence bond and free-energy perturbation simulations, we explore the feasibility of various mechanisms for the deprotonation of the 3'-OH group of the primer DNA strand, and the subsequent formation and cleavage of P-O bonds in four Polβ, two truncated Polλ (tPolλ), and two tPolλ Loop1 mutant (tPolλΔL1) systems differing in the initial X-ray crystal structure and nascent base pair. The average calculated activation free energies of 14, 18, and 22 kcal mol for Polβ, tPolλ, and tPolλΔL1, respectively, reproduce the trend in the observed catalytic rate constants. The most feasible reaction pathway consists of two successive steps: specific base (SB) proton transfer followed by rate-limiting concerted formation and cleavage of the P-O bonds. We identify linear free-energy relationships (LFERs) which show that the differences in the overall activation and reaction free energies among the eight studied systems are determined by the reaction free energy of the SB proton transfer. We discuss the implications of the LFERs and suggest pK of the 3'-OH group as a predictor of the catalytic rate of X-family DNA polymerases.
人类X家族DNA聚合酶β（Polβ）和λ（Polλ）在细胞DNA损伤反应的碱基切除修复途径中催化核苷酸转移反应。我们使用经验价键和自由能微扰模拟，探讨了引物DNA链3'-OH基团去质子化的各种机制的可行性，以及在四个Polβ、两个截短的Polλ（tPolλ）和两个tPolλ Loop1突变体（tPolλΔL1）系统中P-O键的后续形成和断裂，这些系统在初始X射线晶体结构和新生碱基对上存在差异。Polβ、tPolλ和tPolλΔL1计算得到的平均活化自由能分别为14、18和22 kcal/mol，重现了观察到的催化速率常数的趋势。最可行的反应途径包括两个连续步骤：特定碱基（SB）质子转移，随后是限速的P-O键协同形成和断裂。我们确定了线性自由能关系（LFERs），表明所研究的八个系统之间的整体活化和反应自由能差异由SB质子转移的反应自由能决定。我们讨论了LFERs的意义，并提出3'-OH基团的pK作为X家族DNA聚合酶催化速率的预测指标。
