Reaction mechanism of the epsilon subunit of E. coli DNA polymerase III: insights into active site metal coordination and catalytically significant residues.
Reaction mechanism of the epsilon subunit of E. coli DNA polymerase III: insights into active site metal coordination and catalytically significant residues.
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DOI：
10.1021/ja8082818
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发表时间：
2009-02-04
影响因子：
15
通讯作者：
Darden TA
中科院分区：
文献类型：
--
作者：
Cisneros GA;Perera L;Schaaper RM;Pedersen LC;London RE;Pedersen LG;Darden TA
大肠杆菌DNA聚合酶III的28kDa ε亚基是负责编辑聚合酶插入错误的核外核苷酸校对器。在这里，我们研究了ε进行外切酶活性的机制。我们对含有外切酶活性的n端结构域进行了量子力学/分子力学计算。研究了自由-ε和与θ同源物结合的配合物（HOT）。对于ε-HOT配合物，研究了Mg2+或Mn2+作为必需的二价金属辅因子，而游离-ε仅使用Mg2+。在所有的计算中，与催化金属结合的水分子充当了磷酸键水解的亲核试剂。最初，观察到质子直接转移到H162。随后，发生亲核攻击，接着是第二次质子转移到E14。结果表明，用Mn2+催化的反应速度比用Mg2+催化的反应速度快，与实验结果一致。此外，ε-HOT配合物的能垒略低于自由-ε。在所有情况下，催化金属都是五配位的。电荷和前沿轨道分析表明，电荷转移可以稳定五配位。通过能量分解分析研究各残基对催化作用的贡献，发现有几个重要残基。其中，H98、D103、D129和D146在诱变研究中具有催化作用。其中一些残基在人类TREX1、大肠杆菌DNA - pol I的核酸外切酶结构域和噬菌体RB69的DNA聚合酶上具有结构保守性。
The 28kDa ε subunit of Escherichia coli DNA polymerase III is the exonucleotidic proofreader responsible for editing polymerase insertion errors. Here, we study the mechanism by which ε carries out the exonuclease activity. We performed quantum mechanics/molecular mechanics calculations on the N–terminal domain containing the exonuclease activity. Both the free–ε and a complex, ε bound to a θ homolog (HOT), were studied. For the ε–HOT complex, Mg2+ or Mn2+ were investigated as the essential divalent metal cofactors, while only Mg2+ was used for free–ε. In all calculations, a water molecule bound to the catalytic metal acts as the nucleophile for the hydrolysis of the phosphate bond. Initially, a direct proton transfer to H162 is observed. Subsequently, the nucleophilic attack takes place, followed by a second proton transfer to E14. Our results show that the reaction catalyzed with Mn2+ is faster than with Mg2+, in agreement with experiment. In addition, the ε–HOT complex shows a slightly lower energy barrier compared to free–ε. In all cases the catalytic metal is observed to be penta–coordinated. Charge and frontier orbital analyses suggest that charge transfer may stabilize the penta–coordination. Energy decomposition analysis to study the contribution of each residue to catalysis suggests that there are several important residues. Among these, H98, D103, D129 and D146 have been implicated in catalysis by mutagenesis studies. Some of these residues were found to be structurally conserved on human TREX1, the exonuclease domains from E. coli DNA–Pol I, and the DNA polymerase of bacteriophage RB69.
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