Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: Implications for the catalytic mechanism
Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: Implications for the catalytic mechanism
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DOI：
10.1006/jmbi.2001.4529
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发表时间：
2001-04-06
影响因子：
5.6
通讯作者：
Rupp, B
中科院分区：
文献类型：
--
作者：
Beernink, PT;Segelke, BW;Rupp, B
人类主要的碱性核酸内切酶APE1是一种重要的DNA修复酶，它能去除DNA中的无嘌呤/脱嘧啶碱基，去除3‘复制阻断部分，并调节几种转录调节因子的DNA结合活性。我们测定了全长人APE1酶的两种新晶型的X射线结构，一种是中性的，另一种是酸性的。新结构与先前确定的截短APE1蛋白的结构相似，但在几个环区的构象和弱电子密度的残基跨度上有所不同。在低pH下测得的结构中只有一个活性中心的金属离子，而在APE1核酸酶活性的最适pH值(pH 7.5)下生长的晶体中，活性中心有两个金属离子，两个金属离子的结合间隔为5埃。酶动力学数据表明，至少有两个金属结合位点在功能上是重要的，因为钙离子对依赖于镁离子的APE1的催化表现出复杂的刺激和抑制作用，尽管钙离子本身并不是辅因子。综上所述，结构和动力学数据表明，APE1通过两种金属离子介导的机制催化DNA骨架的水解。(C)2001年学术出版社。
The major human abasic endonuclease, Ape1, is an essential DNA repair enzyme that initiates the removal of apurinic/apyrimidinic sites from DNA, excises 3' replication-blocking moieties, and modulates the DNA binding activity of several transcriptional regulators. We have determined the X-ray structure of the full-length human Ape1 enzyme in two new crystal forms, one at neutral and one at acidic pH. The new structures are generally similar to the previously determined structure of a truncated Ape1 protein, but differ in the conformation of several loop regions and in spans of residues with weak electron density. While only one active-site metal ion is present in the structure determined at low pH, the structure determined from a crystal grown at the pH optimum of Ape1 nuclease activity, pH 7.5, has two metal ions bound 5 Angstrom apart in the active site. Enzyme kinetic data indicate that at least two metal-binding sites are functionally important, since Ca2+ exhibits complex stimulatory and inhibitory effects on the Mg2+-dependent catalysis of Ape1, even though Ca2+ itself does not serve as a cofactor. In conjunction, the structural and kinetic data suggest that Ape1 catalyzes hydrolysis of the DNA backbone through a two metal ion-mediated mechanism. (C) 2001 Academic Press.