The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pKa
The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pKa
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DOI：
10.1261/rna.1102308
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发表时间：
2008-09-01
期刊：
影响因子：
4.5
通讯作者：
Collins, Richard A.
中科院分区：
文献类型：
--
作者：
Smith, M. Duane;Mehdizadeh, Reza;Collins, Richard A.
一些小的核酶使用一般的酸碱催化作为一种机制来促进位点特异性的RNA切割，即使RNA的核糖核苷构建块上的官能团的pK(A)值远离生理pH。裂解反应的速率受到反应溶液中金属阳离子的同一性的强烈影响，但不同阳离子促进速率的机理(S)尚未确定。使用脉孢菌和核酶，我们提供了证据，不同的阳离子赋予催化核苷酸碱基的表观pK(A)值特定的位移，这反过来又决定了在给定的pH下适合一般酸碱催化的质子化状态的RNA的比例，这决定了观察到的切割反应的速度。尽管观察到的不同阳离子的裂解速率有很大差异，但一般酸碱催化的数学模型表明，断裂步骤的本征速率k(1)基本上是恒定的，与反应溶液中阳离子(S)的同一性无关。因此，与在核酶化学机制中调用金属离子的独特作用的模型相反，我们发现VS核酶中的大多数甚至所有的离子特异性速率增强可以完全由离子对核苷酸碱基PK(A)的影响来解释。K(1)本质上是常数的推论表明，动力学模糊问题的解决有利于一个模型，在该模型中，较低的Pk(A)是一般酸的Pk(A)，较高的Pk(A)是一般碱的Pk(A)。
Several small ribozymes employ general acid-base catalysis as a mechanism to enhance site-specific RNA cleavage, even though the functional groups on the ribonucleoside building blocks of RNA have pK(a) values far removed from physiological pH. The rate of the cleavage reaction is strongly affected by the identity of the metal cation present in the reaction solution; however, the mechanism(s) by which different cations contribute to rate enhancement has not been determined. Using the Neurospora VS ribozyme, we provide evidence that different cations confer particular shifts in the apparent pK(a) values of the catalytic nucleobases, which in turn determines the fraction of RNA in the protonation state competent for general acid-base catalysis at a given pH, which determines the observed rate of the cleavage reaction. Despite large differences in observed rates of cleavage in different cations, mathematical models of general acid-base catalysis indicate that k(1), the intrinsic rate of the bond-breaking step, is essentially constant irrespective of the identity of the cation(s) in the reaction solution. Thus, in contrast to models that invoke unique roles for metal ions in ribozyme chemical mechanisms, we find that most, and possibly all, of the ion-specific rate enhancement in the VS ribozyme can be explained solely by the effect of the ions on nucleobase pk(a). The inference that k(1) is essentially constant suggests a resolution of the problem of kinetic ambiguity in favor of a model in which the lower pK(a) is that of the general acid and the higher pk(a) is that of the general base.