Catalytic Mechanism of Human Ten-Eleven Translocation-2 (TET2) Enzyme: Effects of Conformational Changes, Electric Field, and Mutations
Catalytic Mechanism of Human Ten-Eleven Translocation-2 (TET2) Enzyme: Effects of Conformational Changes, Electric Field, and Mutations
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DOI：
10.1021/acscatal.0c05034
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发表时间：
2021-03-14
期刊：
影响因子：
12.9
通讯作者：
Christov, Christo Z.
中科院分区：
文献类型：
--
作者：
Waheed, Sodiq O.;Chaturvedi, Shobhit S.;Christov, Christo Z.
10-11 易位 (TET) 家族酶是非血红素 Fe(II) 和 2-酮戊二酸 (2OG) 依赖性加氧酶，可氧化 DNA 上 5-甲基胞嘧啶 (5mC) 的甲基。 TET 酶在表观遗传修饰中发挥着至关重要的作用，并与恶性转化和各种形式的癌症（如前列腺癌、肺癌和乳腺癌）有关。在本研究中，采用分子动力学（MD）和组合量子力学/分子力学（QM/MM）方法来探索人TET2酶从5mC底物第一次氧化为5hmC期间的催化机制、构象动力学和突变的影响。研究表明，TET2 中的主要结构元件、甘氨酸-丝氨酸 (GS) 连接体和富含 Cys 的 N 末端 (Cys-N) 亚结构域之间的相关运动在野生型 (WT) TET2 中 DNA 底物的方向中起着关键作用。这种相关运动在 TET2 突变体中受到影响。 WT TET2 中的构象变化影响氢原子提取 (HAT) 步骤的速率；然而，其通过 s 通道的机制保持不变。这些结果使我们能够识别对 HAT 至关重要的关键残基，并描述它们的关键能量贡献和长程相关相互作用。值得注意的是，远离 TET2 酶活性位点的几个远程突变出乎意料地对 HAT 步骤产生了重大影响，通过 (i) 增加所需的激活势垒和 (ii) 将电子转移机制从 s 通道切换到 p 通道。值得注意的是，突变改变了沿 Fe.O 键的内部电场，与几何因素（例如夺氢距离和角度）的变化协同影响 TET2 突变体形式的反应性。动力学同位素效应 (KIE) 计算表明 WT 中的隧道效应较弱，且突变体形式存在差异。双突变形式 K1299E-S1303N 对难治性贫血患者具有临床意义，对激活屏障、电场和 KIE 具有显着影响。这项研究为人类 TET2 酶的分子生物物理学和病理学提供了新的见解，并断言了第二个领域及其他领域的蛋白质残基对催化过程的重要影响。
Ten-eleven translocation (TET) family of enzymes are non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases that perform oxidation of the methyl group of the 5-methylcytosine (5mC) on DNA. TET enzymes play a crucial role in epigenetic modifications and have been linked to malignant transformation and various forms of cancer such as prostate, lung, and breast cancer. In this study, molecular dynamic (MD) and combined quantum mechanic/molecular mechanic (QM/MM) approaches were used to explore the catalytic mechanism, conformational dynamics, and the effects of mutations during the first oxidation from 5mC substrate to 5hmC by human TET2 enzyme. The studies reveal that a correlated motion between the main structural elements in TET2, the glycine-serine (GS) linker and the Cys-rich Nterminal (Cys-N) subdomain, plays a key role in the orientation of the DNA substrate in the wild-type (WT) TET2. This correlated motion is affected in the mutant forms of TET2. The conformational changes in the WT TET2 influence the rate of the hydrogen atom abstraction (HAT) step; however, its mechanism via s-channel remains unchanged. The results enabled us to identify key residues that are crucial for HAT and to delineate their crucial energy contributions and long-range correlated interactions. Notably, several remote mutations, far away from the TET2 enzymes' active site, unexpectedly exercise a substantial effect on the HAT step by (i) increasing the required activation barrier and (ii) switching the electron transfer mechanism from s- to p-channel. Remarkably, mutations alter the internal electric fields along the Fe.O bond that in synergy with changes in the geometric factors (e.g., the hydrogen abstraction distance and the angle) influence the reactivity of the TET2 mutant forms. The kinetic isotope effect (KIE) calculations indicate weak tunneling contributions in the WT, with variations in the mutant forms. The double-mutant form K1299E-S1303N, which has clinical implications in patients with refractory anemia, exercises a substantial effect on the activation barrier, electric field, and the KIE. This study offers a novel insight into molecular biophysics and pathology of the human TET2 enzyme and asserts the vital effects of the protein residues in the second sphere and beyond on the catalytic process.