Kinetic mechanism for the excision of hypoxanthine by Escherichia coli AlkA and evidence for binding to DNA ends.
Kinetic mechanism for the excision of hypoxanthine by Escherichia coli AlkA and evidence for binding to DNA ends.
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DOI：
10.1021/bi200232c
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发表时间：
2011-05-24
期刊：
影响因子：
2.9
通讯作者：
O'Brien PJ
中科院分区：
文献类型：
--
作者：
Zhao B;O'Brien PJ
大肠杆菌 3-甲基腺嘌呤 DNA 糖基化酶 II 蛋白 (AlkA) 可识别多种氧化和烷基化碱基损伤，并催化 N-糖苷键水解，启动碱基切除修复途径。尽管该酶是 25 多年前发现的首批 DNA 修复糖基化酶之一，并且具有多种晶体结构，但其机制仍知之甚少。因此，我们表征了 AlkA 催化的脱氨基嘌呤、次黄嘌呤切除的动力学机制。多次周转糖基化酶测定与 Michaelis-Menten 动力学一致。然而，在通常用于研究其他 DNA 糖基化酶的单周转条件下，我们观察到不寻常的双相蛋白质饱和曲线。最初，观察到的切除速率常数随着 AlkA 蛋白的增加而增加，但在蛋白质浓度较高时，速率常数降低。这种行为最容易解释为与 DNA 末端的紧密结合以及 DNA 上多个 AlkA 原聚体的拥挤。与该模型一致，晶体结构显示 AlkA 优先结合 DNA 末端。通过改变病变的位置，我们鉴定了一种不对称底物，该底物在较高浓度的 AlkA 下不表现出抑制作用，并且我们进行了稳态前和稳态动力学分析。与其他糖基化酶不同，无碱基产物的释放比 N-糖苷键裂解更快。然而，AlkA 在多次周转条件下表现出显着的产物抑制，并且它与脱碱基位点的结合比与次黄嘌呤损伤位点的结合紧密约 10 倍。当对 DNA 烷基化的适应性反应被激活并且存在非常高水平的 AlkA 蛋白时，这种紧密结合有助于保护脱碱基位点。
The Escherichia coli 3-methyladenine DNA glycosylase II protein (AlkA) recognizes a broad range of oxidized and alkylated base lesions and catalyzes the hydrolysis of the N-glycosidic bond to initiate the base excision repair pathway. Although the enzyme was one of the first DNA repair glycosylases to be discovered more than 25 years ago, and there are multiple crystal structures, the mechanism is poorly understood. Therefore, we have characterized the kinetic mechanism for the AlkA-catalyzed excision of the deaminated purine, hypoxanthine. The multiple turnover glycosylase assays are consistent with Michaelis-Menten kinetics. However, under single turnover conditions that are commonly employed to study other DNA glycosylases, we observe an unusual biphasic protein saturation curve. Initially the observed rate constant for excision increases with increasing AlkA protein, but at higher concentrations of protein the rate constant decreases. This behavior can be most easily explained by tight binding to DNA ends and by crowding of multiple AlkA protamers on the DNA. Consistent with this model, crystal structures have shown the preferential binding of AlkA to DNA ends. By varying the position of the lesion, we identified an asymmetric substrate that does not show inhibition at higher concentrations of AlkA and we performed pre-steady state and steady state kinetic analysis. Unlike other glycosylases, release of the abasic product is faster than N-glycosidic bond cleavage. Nevertheless, AlkA exhibits significant product inhibition under multiple-turnover conditions, and it binds approximately 10-fold more tightly to an abasic site than to a hypoxanthine lesion site. This tight binding could help protect abasic sites when the adaptive response to DNA alkylation is activated and very high levels of AlkA protein are present.
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