Amino Acid Substitution in the Active Site of DNA Polymerase β Explains the Energy Barrier of the Nucleotidyl Transfer Reaction
Amino Acid Substitution in the Active Site of DNA Polymerase β Explains the Energy Barrier of the Nucleotidyl Transfer Reaction
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DOI：
10.1021/ja403842j
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发表时间：
2013-05-29
影响因子：
15
通讯作者：
Wilson, Samuel H.
中科院分区：
文献类型：
--
作者：
Batra, Vinod K.;Perera, Lalith;Wilson, Samuel H.
DNA 聚合酶 beta (pol beta) 是一种双功能酶，因其在碱基切除 DNA 修复中的作用而被广泛研究，其中一个关键功能是间隙填充 DNA 合成。尽管近年来取得了重大进展，但 DNA 合成反应的原子级机制仍然知之甚少。基于pol beta与其底物复合物的晶体结构以及活性位点中氨基酸和金属的理论考虑，我们提出Asp256附近的羧酸根基团通过接受来自引物O3'基团的质子来实现反应，从而激活O3'作为反应路径中的亲核试剂。在这里，我们通过将 Asp256 的侧链更改为 Glu 来测试该提议，然后探讨这种保守的变化对反应的影响。 D256E酶的活性比野生型酶低1000倍以上，并且D256E和野生型酶的活性位点的晶体结构略有不同。 D256E 酶 DNA 合成的理论分析表明，O3' 质子仍然转移到残基 256 附近的羧酸盐。然而，与野生型相比，反应路径中 O3' 质子转移的静电稳定性和位置发生了显着改变。令人惊讶的是，这是由于 Glu256 酶活性位点中 Arg254 侧链的重新定位，使得 Arg254 无法稳定来自 O3' 的质子转移。野生型酶的理论结果表明与 O3' 质子转移相关的早期电荷重组，而这在 D256E 酶中不会发生。电荷重组是由活性位点中的催化镁离子介导的。
DNA polymerase beta (pol beta) is a bifunctional enzyme widely studied for its roles in base excision DNA repair, where one key function is gap filling DNA synthesis. In spite of significant progress in recent years, the atomic level mechanism of the DNA synthesis reaction has remained poorly understood. Based on crystal structures of pol beta in complex with its substrates and theoretical considerations of amino acids and metals in the active site, we have proposed that a nearby carboxylate group of Asp256 enables the reaction by accepting a proton from the primer O3'group, thus activating O3'as the nucleophile in the reaction path. Here, we tested this proposal by altering the side chain of Asp256 to Glu and then exploring the impact of this conservative change on the reaction. The D256E enzyme is more than 1000 fold less active than the wild type enzyme, and the crystal structures are subtly different in the active sites of the D256E and wild type enzymes. Theoretical analysis of DNA synthesis by the D256E enzyme shows that the O3'proton still transfers to the nearby carboxylate of residue 256. However, the electrostatic stabilization and location of the O3' proton transfer during the reaction path are dramatically altered compared with wild type Surprisingly, this is due to repositioning of the Arg254 side chain in the Glu256 enzyme active site, such that Arg254 is not in position to stabilize the proton transfer from O3'. The theoretical results with the wild type enzyme indicate an early charge reorganization associated with the O3' proton transfer, and this does not occur in the D256E enzyme. The charge reorganization is mediated by the catalytic magnesium ion in the active site.