One- and two-metal ion catalysis: global single-turnover kinetic analysis of the PvuII endonuclease mechanism.
One- and two-metal ion catalysis: global single-turnover kinetic analysis of the PvuII endonuclease mechanism.
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DOI：
10.1021/bi801027k
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发表时间：
2008-11-25
期刊：
影响因子：
2.9
通讯作者：
Dupureur, Cynthia M.
中科院分区：
文献类型：
--
作者：
Xie, Fuqian;Qureshi, Shabir H.;Papadakos, Grigorios A.;Dupureur, Cynthia M.
酯水解是生物化学中最普遍的反应之一。许多这些反应依赖于金属离子的各种机械步骤。大量金属依赖性核酸酶已结晶，其活性位点具有两个金属离子。尽管这些金属离子在核酸水解中的作用正在进行讨论，但很少有研究使用天然辅因子Mg（II）和反应进程曲线的全局拟合来研究这个问题。作为代表性的同源二聚体PvuII核酸内切酶的全面研究的一部分，我们已经收集了单周转DNA切割数据作为Mg（II）浓度的函数，并在全球范围内拟合这些数据的一些模型，测试金属核酸酶机制的各个方面。DNA缔合速率常数是约100倍高的催化非支持性的Ca（II）与天然辅因子Mg（II）的存在下，突出了一个有趣的辅因子的差异。其中金属离子先于DNA结合的途径在动力学上是有利的。数据拟合良好的模型，其中一个和两个金属离子每个活性位点（EM2S和EM4S）支持裂解。有趣的是，EM2S的裂解速率比EM4S慢约100倍。总的来说，这些数据表明，对于PvuII系统，涉及每个活性位点一个金属离子的催化确实可以发生，但是在饱和金属离子（体外）条件下可以操作更有效的两个金属离子机制。
Ester hydrolysis is one of the most ubiquitous reactions in biochemistry. Many of these reactions rely on metal ions for various mechanistic steps. A large number of metal dependent nucleases have been crystallized with two metal ions in their active sites. In spite of an ongoing discussion about the roles of these metal ions in nucleic acid hydrolysis, there are very few studies which examine this issue using the native cofactor Mg(II) and global fitting of reaction progress curves. As part of a comprehensive study of the representative homodimeric PvuII endonuclease, we have collected single turnover DNA cleavage data as a function of Mg(II) concentration and globally fit these data to a number of models which test various aspects of metallonuclease mechanism. DNA association rate constants are about 100-fold higher in the presence of the catalytically nonsupportive Ca(II) vs. the native cofactor Mg(II), highlighting an interesting cofactor difference. A pathway in which metal ions bind prior to DNA is kinetically favored. The data fit well to a model in which both one and two metal ions per active site (EM2S and EM4S) support cleavage. Interestingly, the cleavage rate for EM2S is about 100-fold slower than that displayed by EM4S. Collectively, these data indicate that for the PvuII system, catalysis involving one metal ion per active site can indeed occur, but that a more efficient two metal ion mechanism can be operative under saturating metal ion (in vitro) conditions.
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