Ca+ binding in the active site of HincII:: Implications for the catalytic mechanism
Ca+ binding in the active site of HincII:: Implications for the catalytic mechanism
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DOI：
10.1021/bi0490082
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发表时间：
2004-10-26
期刊：
影响因子：
2.9
通讯作者：
Horton, NC
中科院分区：
文献类型：
--
作者：
Etzkorn, C;Horton, NC
给出了与Ca 2+和含有GTCGAC的同源DNA结合的II型限制性内切酶HincII的2.8埃晶体结构。该DNA是未切割的，并且每个活性位点结合一个钙离子，在先前描述为相关平切II型限制性内切核酸酶EcoRV中的位点I的位置[Horton，N. C.的方法，纽贝里，K. J.，和Perona，J.J.（1998）Proc.Natl. Acad. Sci. U.S.A. 95（23），13489-13494]，以及在其它相关酶中发现的那些。与EcoRV中的位点I金属不同，但与PvuII、NgoMIV、BamHI、Bgl I和Bgl I的位点I金属类似，观察到的钙阳离子直接连接到易裂磷酸盐的pro-S-p氧。钙离子连接的水分子在磷酸二酯键裂解反应中很好地定位为亲核试剂，并且在保守的活性位点赖氨酸（Lys 129）的氢键距离内，以及易断裂的磷酸盐的磷酸基团3'的pro-R-p氧，表明这些基团在催化机制中可能的作用。动力学数据与HincII在DNA切割中的3 '-磷酸基团的重要作用一致。先前观察到的钠离子[Horton，N. C.的方法，多纳湖F.、和Perona，J. J.（2002）Nat.Struct.Biol.9，42-47]在Ca 2+结合结构的活性位点中持续存在;然而，动力学数据显示在不存在Na+离子的情况下对DNA切割的单周转率几乎没有影响。
The 2.8 Angstrom crystal structure of the type II restriction endonuclease HincII bound to Ca2+ and cognate DNA containing GTCGAC is presented. The DNA is uncleaved, and one calcium ion is bound per active site, in a position previously described as site I in the related blunt cutting type II restriction endonuclease EcoRV [Horton, N. C., Newberry, K. J., and Perona, J. J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95 (23), 13489-13494], as well as that found in other related enzymes. Unlike the site I metal in EcoRV, but similar to that of PvuII, NgoMIV, BamHI, BglI, and Bgll, the observed calcium cation is directly ligated to the pro-S-p oxygen of the scissile phosphate. A calcium ion-ligated water molecule is well positioned to act as the nucleophile in the phosphodiester bond cleavage reaction, and is within hydrogen bonding distance of the conserved active site lysine (Lys 129), as well as the pro-R-p oxygen of the phosphate group 3' of the scissile phosphate, suggesting possible roles for these groups in the catalytic mechanism. Kinetic data consistent with an important role for the 3'-phosphate group in DNA cleavage by HincII are presented. The previously observed sodium ion [Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47] persists in the active sites of the Ca2+-bound structure; however, kinetic data show little effect on the single-turnover rate of DNA cleavage in the absence of Na+ ions.