Role of an active site adenine in hairpin ribozyme catalysis
Role of an active site adenine in hairpin ribozyme catalysis
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DOI：
10.1016/j.jmb.2005.04.005
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发表时间：
2005-06-24
影响因子：
5.6
通讯作者：
Fedor, MJ
中科院分区：
文献类型：
--
作者：
Kuzmin, YI;Da Costa, CP;Fedor, MJ
发夹状核酶是一种小的催化RNA，可以加速磷酸二酯键的可逆断裂。结构和机理研究表明，二价金属稳定了功能结构，但不直接参与催化。相反，两个活性部位的碱基G8和A38似乎参与了催化化学。通过比较核酶突变体和A38修饰物的切割和连接反应，研究了A38在活性部位结构和化学上的重要特征。A38的碱基替换使切割和连接活性分别降低了14,000倍和37,000倍，突显了该核苷酸碱基在核酶功能中的关键作用。未经修饰的核酶的切割和连接活性随pH的升高而增加，这表明表观pK(A)值接近6的某些官能团的去质子化对活性是重要的。当A38被碱性残基或具有很高或很低pK(A)值的碱基类似物取代时，依赖于pH的活性转变在基本方向上移动了几个pH单位，预计在整个实验pH范围内保持相同的质子化状态。某些外源碱基共享腺嘌呤的酰胺基，使缺乏A38的碱性核酶变异体恢复活性。化学救援反应的pH依赖性也随着救援碱基的内在碱性而改变，进一步证明了嘌呤类似物NI位的质子化状态对救援活性是重要的。这些结果与发夹状核酶催化机理的模型是一致的，在该机理中，与A38的相互作用为过渡态提供了静电稳定。(C)2005爱思唯尔有限公司。保留所有权利。
The hairpin ribozyme is a small catalytic RNA that accelerates reversible cleavage of a phosphodiester bond. Structural and mechanistic studies suggest that divalent metals stabilize the functional structure but do not participate directly in catalysis. Instead, two active site nucleobases, G8 and A38, appear to participate in catalytic chemistry. The features of A38 that are important for active site structure and chemistry were investigated by comparing cleavage and ligation reactions of ribozyme variants with A38 modifications. An abasic substitution of A38 reduced cleavage and ligation activity by 14,000-fold and 370,000-fold, respectively, highlighting the critical role of this nucleobase in ribozyme function. Cleavage and ligation activity of unmodified ribozymes increased with increasing pH, evidence that deprotonation of some functional group with an apparent pK(a) value near 6 is important for activity. The pH-dependent transition in activity shifted by several pH units in the basic direction when A38 was substituted with an abasic residue, or with nucleobase analogs with very high or low pK(a) values that are expected to retain the same protonation state throughout the experimental pH range. Certain exogenous nucleobases that share the amidine group of adenine restored activity to abasic ribozyme variants that lack A38. The pH dependence of chemical rescue reactions also changed according to the intrinsic basicity of the rescuing nucleobase, providing further evidence that the protonation state of the NI position of purine analogs is important for rescue activity. These results are consistent with models of the hairpin ribozyme catalytic mechanism in which interactions with A38 provide electrostatic stabilization to the transition state. (c) 2005 Elsevier Ltd. All rights reserved.