QF / MM MD模拟人类皮瓣核酸内切酶（hFEN1）的酶途径，阐明了常见的RNase H酶裂解途径。,ACS Catalysis

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QF / MM MD模拟人类皮瓣核酸内切酶（hFEN1）的酶途径，阐明了常见的RNase H酶裂解途径。
ACS Catalysis ( IF 11.3 ) Pub Date : 2015-05-26 00:00:00 , DOI: 10.1021/acscatal.5b00178
Jacopo Sgrignani ₁ , Alessandra Magistrato ₂

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襟翼核酸内切酶（FEN）是核酸水解酶，负责切除在滞后链DNA复制或长补丁碱基切除修复（LP-BER）过程中从核酸结构突出的5'-小DNA和RNA片段（襟翼）。在这项工作中，我们首次报告了由人FEN1促进的酶催化的原子性和能量性渲染。重建反应性hFEN /双链（ds）DNA加合物后，我们采用了混合量子经典（QM / MM）动力学和伞形自由能计算，并用AM1 / d-PhoT哈密顿量处理了QM部分，从而完成了酶促循环潜在的所有可能反应途径的广泛表征。易裂磷酸侧翼的核碱基的R p氧。此步骤需要与实验数据一致的自由能量势垒（Δ ģ ^⧧_EXP = 16.1千卡/摩尔VSΔ ˚F ^⧧_计算值= 16±2千卡/摩尔）。由于FEN在维持核酸保真度和细胞增殖中的重要作用，对其酶机制的详细理解引起了广泛的兴趣，阐明了用于保存基因组完整性的关键酶生物学过程，并对医学和生物技术应用具有重要意义。

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QM/MM MD Simulations on the Enzymatic Pathway of the Human Flap Endonuclease (hFEN1) Elucidating Common Cleavage Pathways to RNase H Enzymes

Flap endonucleases (FENs) are nucleic acid hydrolyzing enzymes in charge of excising 5′-small DNA and RNA fragments (flaps) protruding from nucleic acid structures during the lagging strand DNA replication or the long-patch base excision repair (LP-BER) processes. In this work we report, for the first time, an atomistic and energetic rendering of the enzymatic catalysis promoted by the human FEN1. After reconstruction of a reactive hFEN/double strand (ds) DNA adduct we employed mixed quantum-classical (QM/MM) metadynamics and umbrella sampling free energy calculations, with the QM part treated with the AM1/d-PhoT Hamiltonian, to perform an extensive characterization of all possible reaction pathways underlying the enzymatic cycle. Our extensive investigation points to a most likely reaction pathway very similar to that recently proposed for ribonuclease H, in which the rate-determining step is the nucleophilic attack of a water to the scissile phosphate, which occurs concomitantly with its activation by the pro-Rp oxygen of the nucleobase flanking the scissile phosphate. This step requires a free energy barrier in good agreement with experimental data (ΔG^⧧_exp = 16.1 kcal/mol vs ΔF^⧧_calc = 16 ± 2 kcal/mol). Due to the important role of FENs in maintaining nucleic acid fidelity and cell proliferation, a detailed understanding of its enzymatic mechanism has broad interest to elucidate a key enzymatic biological process for preserving genome integrity and has implications for medical and biotechnological applications.

更新日期：2015-05-26

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