Luo N, Mehler E, Osman R
Department of Physiology and Biophysics, Mount Sinai School of Medicine of the City University of New York 10029, USA.
Biochemistry. 1999 Jul 20;38(29):9209-20. doi: 10.1021/bi990262h.
The structure of uracil DNA glycosylase (UDG) in complex with a nonamer duplex DNA containing a uracil has been determined only in the product state. The reactant state was constructed by reattaching uracil to the deoxyribose, and both complexes were studied by molecular dynamics simulations. Significant changes in the positions of secondary structural elements in the enzyme are induced by the hydrolysis of the glycosidic bond. The simulations show that the specificity of the uracil pocket in the enzyme is largely retained in both complexes with the exception of Asn-204, which has been identified as a residue that contributes to discrimination between uracil and cytosine. The hydrogen bond between the amide group of Asn-204 and O(4) of uracil is disrupted by fluctuations of the side chain in the reactant state and is replaced by a hydrogen bond to water molecules trapped in the interior of the protein behind the uracil binding pocket. The role of two residues implicated by mutation experiments to be important in catalysis, His-268 and Asp-145, is clarified by the simulations. In the reactant state, His-268 is found 3.45 +/- 0.34 A from the uracil, allowing a water molecule to form a bridge to O(2). The environment in the enzyme raises the pK(a) value of His-268 to 7.1, establishing a protonated residue for assisting in the hydrolysis of the glycosidic bond. In agreement with the crystallographic structure, the DNA backbone retracts after the hydrolysis to allow His-268 to approach the O(2) of uracil with a concomitant release of the bridging water molecule and a reduction in the pK(a) to 5.5, which releases the proton to the product. The side chain of Asp-145 is fully solvated in the reactant state and H-bonded through a water molecule to the 3'-phosphate of uridine. Both the proximity of Asp-145 to the negatively charged phosphate and its pK(a) of 4.4 indicate that it cannot act as a general base catalyst. We propose a mechanism in which the bridging water between Asp-145 and the 3'-phosphate accepts a proton from another water to stabilize the bridge through a hydronium ion as well as to produce the hydroxide anion required for the hydrolytic step. The mechanism is consistent with known experimental data.
尿嘧啶DNA糖基化酶（UDG）与含有尿嘧啶的九聚体双链DNA形成复合物的结构仅在产物状态下得到确定。通过将尿嘧啶重新连接到脱氧核糖上构建反应物状态，并通过分子动力学模拟对这两种复合物进行研究。糖苷键的水解会诱导酶中二级结构元件位置发生显著变化。模拟结果表明，除了Asn-204外，酶中尿嘧啶口袋的特异性在两种复合物中基本得以保留，Asn-204已被确定为有助于区分尿嘧啶和胞嘧啶的一个残基。在反应物状态下，Asn-204酰胺基团与尿嘧啶O(4)之间的氢键因侧链波动而断裂，并被与被困在尿嘧啶结合口袋后方蛋白质内部的水分子形成的氢键所取代。模拟结果阐明了通过突变实验表明在催化中起重要作用的两个残基His-268和Asp-145的作用。在反应物状态下，发现His-268距尿嘧啶3.45±0.34埃，使得一个水分子能够形成与O(2)的桥连。酶中的环境将His-268的pK(a)值提高到7.1，形成一个质子化残基以协助糖苷键的水解。与晶体结构一致，水解后DNA主链回缩，使得His-268能够接近尿嘧啶的O(2)，同时桥连水分子释放，pK(a)降至5.5，质子释放到产物中。Asp-145的侧链在反应物状态下完全被溶剂化，并通过一个水分子与尿苷的3'-磷酸形成氢键。Asp-145与带负电荷的磷酸基团的接近程度及其4.4的pK(a)值表明它不能作为一般碱催化剂。我们提出一种机制，其中Asp-145与3'-磷酸之间的桥连水分子从另一个水分子接受一个质子，通过水合氢离子稳定桥连，同时产生水解步骤所需的氢氧根阴离子。该机制与已知实验数据一致。
