Thomas Jason M, Perrin David M
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, B.C., Canada, V6T 1Z1.
J Am Chem Soc. 2009 Jan 28;131(3):1135-43. doi: 10.1021/ja807790e.
Recent crystallographic and computational studies have provided fresh insights into the catalytic mechanism of the RNA-cleaving hammerhead ribozyme. Based on these findings, specific ribozyme functional groups have been hypothesized to act directly as the general acid and base catalysts, although the catalytic role of divalent metal cations (M(2+)) remains uncertain. We now report a functional characterization of the general acid catalysis mechanism and the role of an M(2+) cofactor therein, for the S. mansoni hammerhead (an "extended" hammerhead ribozyme). We have compared hammerhead cleavage of substrates with natural (ribo-phosphodiester) versus bridging-5'-phosphorothioate scissile linkages, in the contexts of active site mutations and M(2+) substitution. Cleavage of the natural substrate is inhibited by modification of the G8 2'-OH ribozyme residue and depends strongly upon the presence and identity of an M(2+) cofactor; in contrast, cleavage of the bridging-phosphorothioate substrate is conspicuously insensitive to any of these factors. These results imply that (1) both an M(2+) cofactor and the G8 2'-OH play crucial roles in hammerhead general acid catalysis and (2) the M(2+) cofactor does not contribute to general acid catalysis via Lewis acid stabilization of the leaving group. General acid pK(a) perturbation was also demonstrated for both M(2+) substitution and G8 2'-OH modification, which suggests transition state M(2+) coordination of the G8 2'-OH, to lower its pK(a) and improve its ability to transfer a proton to the leaving group. We also report a simple method for synthesizing radiolabeled bridging-5'-phosphorothioate substrates.
最近的晶体学和计算研究为RNA切割型锤头状核酶的催化机制提供了新的见解。基于这些发现，尽管二价金属阳离子（M(2+)）的催化作用仍不确定，但已推测特定的核酶官能团可直接作为广义酸碱催化剂。我们现在报告了曼氏血吸虫锤头状核酶（一种“扩展”的锤头状核酶）的广义酸催化机制及其M(2+)辅助因子的功能特征。我们在活性位点突变和M(2+)替代的背景下，比较了具有天然（核糖磷酸二酯）与桥连-5'-硫代磷酸酯可裂解连接的底物的锤头状切割。天然底物的切割受到G8 2'-OH核酶残基修饰的抑制，并且强烈依赖于M(2+)辅助因子的存在和特性；相比之下，桥连硫代磷酸酯底物的切割对这些因素中的任何一个都明显不敏感。这些结果表明：（1）M(2+)辅助因子和G8 2'-OH在锤头状广义酸催化中都起着关键作用；（2）M(2+)辅助因子不会通过离去基团的路易斯酸稳定作用来促进广义酸催化。对于M(2+)替代和G8 2'-OH修饰，也证明了广义酸pK(a)的扰动，这表明G8 2'-OH的过渡态M(2+)配位，以降低其pK(a)并提高其将质子转移到离去基团的能力。我们还报告了一种合成放射性标记的桥连-5'-硫代磷酸酯底物的简单方法。
