Identification of the catalytic Mg²⁺ ion in the hepatitis delta virus ribozyme.
Identification of the catalytic Mg²⁺ ion in the hepatitis delta virus ribozyme.
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识别肝炎三角洲病毒核酶中的催化mg²⁺离子。
DOI：
10.1021/bi3013092
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发表时间：
2013-01-22
期刊：
影响因子：
2.9
通讯作者：
Golden BL
中科院分区：
文献类型：
--
作者：
Chen J;Ganguly A;Miswan Z;Hammes-Schiffer S;Bevilacqua PC;Golden BL
丁型肝炎病毒核酶利用催化核碱基和二价金属离子催化RNA裂解反应。催化碱C75作为一般酸，其pKa向中性转移。金属离子在这一机制中的作用尚不清楚。最近对裂解前核酶晶体结构的研究发现，Mg2+离子通过其部分水合球与G25•U20反向摆动相互作用。此外，这个Mg2+离子可以直接配位亲核试剂U（-1）的2 ' -羟基，这表明它可以作为路易斯酸促进2 ' -羟基的去质子化。为了测试活性位点Mg2+离子的作用，我们将G25•U20反向摆动替换为A25•C20反向摆动。这一变化被发现显著降低了活性位点的负电位，正如静电计算所支持的那样，这表明活性位点的Mg2+结合可能受到突变的不利影响。A25•C20双突变体的动力学分析和分子动力学表明，该突变体稳定折叠成活性结构。然而，双突变体的pH-rate谱与野生型核酶谱相反，这表明A25•C20双突变体失去了活性位点金属离子。总的来说，这些研究支持一个模型，其中位于G25•U20反向摆动的部分水合Mg2+具有催化作用，可以作为Lewis酸，Brønsted碱，或两者兼而有之，以促进亲核试剂的去质子化。
The hepatitis delta virus ribozyme catalyzes an RNA cleavage reaction using a catalytic nucleobase and a divalent metal ion. The catalytic base, C75, serves as a general acid and has a pKa shifted towards neutrality. Less is known about the role of metal ions in the mechanism. A recent crystal structure of the pre-cleavage ribozyme identified a Mg2+ ion that interacts through its partial hydration sphere with the G25•U20 reverse wobble. In addition, this Mg2+ ion is in position to directly coordinate the nucleophile, the 2’-hydroxyl of U(-1), suggesting it can serve as a Lewis acid to facilitate deprotonation of the 2’-hydroxyl. To test the role of the active site Mg2+ ion, we replaced the G25•U20 reverse wobble with an isosteric A25•C20 reverse wobble. This change was found to significantly reduce the negative potential at the active site, as supported by electrostatics calculations, suggesting that active site Mg2+ binding could be adversely affected by the mutation. Kinetic analysis and molecular dynamics of the A25•C20 double mutant suggest that this variant stably folds into an active structure. However, pH-rate profiles of the double mutant are inverted relative to the profiles for wild-type ribozyme, suggesting that the A25•C20 double mutant has lost the active site metal ion. Overall, these studies support a model wherein the partially hydrated Mg2+ positioned at the G25•U20 reverse wobble is catalytic and could serve as a Lewis acid, a Brønsted base, or both to facilitate deprotonation of the nucleophile.
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影响因子：
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