Activities and relative affinities of divalent metals in unmodified and phosphorothioate-substituted hammerhead ribozymes.
Activities and relative affinities of divalent metals in unmodified and phosphorothioate-substituted hammerhead ribozymes.
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未修饰和硫代磷酸酯取代的锤头核酶中二价金属的活性和相对亲和力。
DOI：
10.1016/s0162-0134(00)00079-9
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发表时间：
2000
影响因子：
3.9
通讯作者：
DeRose,VJ
中科院分区：
文献类型：
--
作者：
Hunsicker,LM;DeRose,VJ
金属在锤头状核酶磷酸二酯键断裂反应中的作用正在研究中。在这项研究中，几种不同的金属支持核酶活性的表观亲和力和能力的报告。通过测量二价阳离子释放结合的Mn 2+的能力来确定二价阳离子对锤头状核酶的相对亲和力。EPR检测的Mn ~（2+）竞争研究表明，Mn ~（2+）> Co ~（2+）> Zn ~（2+）> Cd ~（2+）> Mg ~（2+）。这种排序一般遵循在pH 7.0，0.1 M NaCl和饱和金属浓度下测定的最大裂解速率的趋势，Mn 2 +> Co 2 +> Cd 2 +> Mg 2+。在这些条件下，观察到Mn 2+的最大速率，这可能与该离子的高亲和力、低pKa和低Δ Hhyd有关。将硫代磷酸酯5′取代到酶链中的9个腺苷中的每一个上，导致锤头复合物的Mn 2+结合特性发生变化。在硫代磷酸酯取代的锤头状复合物中，8至9个Mn 2+以两种类型结合：“1型”（n=1±0.3，Kd=1.1±1 μM）和较弱的“2型”（n=7.7±0.3，Kd=125±27 μM）。多个硫代磷酸酯取代导致在未修饰的核酶中观察到的两个至三个较高亲和力位点的损失。硫代磷酸酯取代的核酶的金属竞争研究表明，金属的相对亲和力为Cd ~（2+）> Zn ~（2+）> Co ~（2+），Mg ~（2+），其中Mn ~（2+）被取代的数量和亲硫性Cd ~（2+）的表观亲和力受硫代磷酸酯取代的影响最大。
The roles of metals in the phosphodiester bond cleavage reaction performed by the hammerhead ribozyme are under investigation. In this study, the apparent affinities and the abilities of several different metals to support ribozyme activity are reported. The relative affinities of divalent cations for the hammerhead ribozyme are determined by measuring their ability to release bound Mn2+. The EPR-detected Mn2+competition studies give an order of apparent affinity of Mn2+∼ Co2+∼ Zn2+>Cd2+≫Mg2+. This ordering generally follows the trend of maximum rates of cleavage determined at pH 7.0, 0.1 M NaCl, and saturating metal concentrations, of Mn2+>Co2+>Cd2+>Mg2+. The maximum rate is observed for Mn2+under these conditions and may be related to the high affinity, low pKaand low ΔHhydof this ion. Substitution of phosphorothioates 5′ to each of the nine adenosines in the enzyme strand yields a change in the Mn2+binding properties of the hammerhead complex. In the phosphorothioate-substituted hammerhead complex, eight to nine Mn2+bind in two types of classes: ‘type 1’ (n=1±0.3, Kd=1.1±1 μM) and weaker ‘type 2’ (n=7.7±0.3, Kd=125±27 μM). The multiple phosphorothioate substitutions result in the loss of two to three of the higher affinity sites observed in the unmodified ribozyme. Metal competition studies with the phosphorothioate-substituted ribozyme indicate that the relative affinities of the metals are Cd2+>Zn2+>Co2+, Mg2+with the number of Mn2+displaced and apparent affinity of the thiophilic Cd2+most affected by the phosphorothioate substitutions.