Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10-23
Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10-23
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DOI：
10.1111/j.1742-4658.2007.05655.x
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发表时间：
2007-02-01
期刊：
影响因子：
5.4
通讯作者：
Stec, Wojciech J.
中科院分区：
文献类型：
--
作者：
Nawrot, Barbara;Widera, Kinga;Stec, Wojciech J.
在3 mM Mg 2+或3 mM Mn 2+存在下，在单转换条件下，评价了一系列16种硫代脱氧核酶10-23的RNA磷酸二酯键切割活性，所述硫代脱氧核酶10-23在催化核心的预定位置从P1至P16含有P-立构无规单硫代磷酸酯键。金属特异性开关方法允许鉴定位于核心的七个位置（P2、P4和P9-13）的非桥接磷酸氧（proR（P）或proS（P）），其参与与二价金属离子的直接配位。相比之下，在位置P3、P6、P7和P14-16处的硫代磷酸酯在脱氧核酶介导的催化中没有显示出功能相关性。有趣的是，在位置P1或P8处的硫代磷酸酯修饰增强了酶的催化效率。在所测试的脱氧核酶中，在Mg 2+存在下，在位置P5处的硫代取代对催化速率具有最大的有害影响，并且这在Mn 2+存在下是逆转的。用立体定向的P-手性的硫代脱氧核酶的进一步实验表明，P5磷酸的氧和P9处的proR（P）氧直接参与金属离子配位。此外，还发现G（6）的C6氧原子有助于金属离子的结合，这种相互作用对10-23脱氧核酶的催化活性是必不可少的。
The RNA phosphodiester bond cleavage activity of a series of 16 thio-deoxyribozymes 10-23, containing a P-stereorandom single phosphorothioate linkage in predetermined positions of the catalytic core from P1 to P16, was evaluated under single-turnover conditions in the presence of either 3 mM Mg2+ or 3 mM Mn2+. A metal-specificity switch approach permitted the identification of nonbridging phosphate oxygens (proR(P) or proS(P)) located at seven positions of the core (P2, P4 and P9-13) involved in direct coordination with a divalent metal ion(s). By contrast, phosphorothioates at positions P3, P6, P7 and P14-16 displayed no functional relevance in the deoxyribozyme-mediated catalysis. Interestingly, phosphorothioate modifications at positions P1 or P8 enhanced the catalytic efficiency of the enzyme. Among the tested deoxyribozymes, thio-substitution at position P5 had the largest deleterious effect on the catalytic rate in the presence of Mg2+, and this was reversed in the presence of Mn2+. Further experiments with thio-deoxyribozymes of stereodefined P-chirality suggested direct involvement of both oxygens of the P5 phosphate and the proR(P) oxygen at P9 in the metal ion coordination. In addition, it was found that the oxygen atom at C6 of G(6) contributes to metal ion binding and that this interaction is essential for 10-23 deoxyribozyme catalytic activity.