Uracil-DNA glycosylase acts by substrate autocatalysis
Uracil-DNA glycosylase acts by substrate autocatalysis
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DOI：
10.1038/35099587
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发表时间：
2001-10-18
期刊：
影响因子：
64.8
通讯作者：
Karplus, M
中科院分区：
文献类型：
--
作者：
Dinner, AR;Blackburn, GM;Karplus, M
在人类中，由于脱氧尿苷（dU）的错误掺入或胞嘧啶的脱氨作用，每天每个细胞的DNA中都会出现数百个尿嘧啶碱基。在人类基因组中已鉴定出四种酶，它们催化DNA中dU的糖苷键水解，产生无嘧啶位点，这是碱基切除修复的第一步（1）。这些尿嘧啶 - DNA糖基化酶中最有效且特性明确的是UDG（也称为UNG，几乎存在于所有已知生物中）（2），它能从单链或双链DNA中切除U，并与DNA复制叉相关（3）。我们使用混合量子力学/分子力学（QM/MM）方法（4）来确定UDG的催化机制。与最初提出的协同结合机制（5 - 10）相反，我们在此表明该反应以逐步解离的方式进行（11,12）。糖苷键的断裂产生一种包含氧鎓阳离子和尿嘧啶阴离子的中间体。随后水分子的攻击以及质子向D145的转移产生了产物。令人惊讶的是，降低活化能的主要贡献来自底物，而非酶。这种“自催化”源于四个磷酸基团的掩埋和定位，它们稳定了决定反应速率的过渡态。这些磷酸基团的重要性解释了缺乏关键残基的突变体所观察到的残留活性（6 - 9）。相应的催化机制可能适用于TDG和SMUG1这两种DNA糖基化酶，它们与UDG属于同一结构超家族（13,14）。
In humans, uracil appears in DNA at the rate of several hundred bases per cell each day as a result of misincorporation of deoxyuridine (dU) or deamination of cytosine. Four enzymes that catalyse the hydrolysis of the glycosylic bond of dU in DNA to yield an apyridiminic site as the first step in base excision repair have been identified in the human genome(1). The most efficient and well characterized of these uracil-DNA glycosylases is UDG (also known as UNG and present in almost all known organisms)(2), which excises U from single- or double-stranded DNA and is associated with DNA replication forks(3). We used a hybrid quantum-mechanical/molecular-mechanical (QM/MM) approach(4) to determine the mechanism of catalysis by UDG. In contrast to the concerted associative mechanism proposed initially (5-10), we show here that the reaction proceeds in a stepwise dissociative manner(11,12). Cleavage of the glycosylic bond yields an intermediate comprising an oxocarbenium cation and a uracilate anion. Subsequent attack by a water molecule and transfer of a proton to D145 result in the products. Surprisingly, the primary contribution to lowering the activation energy comes from the substrate, rather than from the enzyme. This 'autocatalysis' derives from the burial and positioning of four phosphate groups that stabilize the rate-determining transition state. The importance of these phosphates explains the residual activity observed for mutants that lack key residues(6-9). A corresponding catalytic mechanism could apply to the DNA glycosylases TDG and SMUG1, which belong to the same structural superfamily as UDG(13,14).