Kuzmin Yaroslav I, Da Costa Carla P, Cottrell Joseph W, Fedor Martha J
Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
J Mol Biol. 2005 Jun 24;349(5):989-1010. doi: 10.1016/j.jmb.2005.04.005. Epub 2005 Apr 20.
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 N1 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.
发夹状核酶是一种小的催化RNA,可加速磷酸二酯键的可逆切割。结构和机制研究表明,二价金属稳定功能结构,但不直接参与催化。相反,两个活性位点核碱基G8和A38似乎参与催化化学过程。通过比较具有A38修饰的核酶变体的切割和连接反应,研究了对活性位点结构和化学过程重要的A38的特征。A38的无碱基取代分别使切割和连接活性降低了14000倍和370000倍,突出了该核碱基在核酶功能中的关键作用。未修饰核酶的切割和连接活性随pH值升高而增加,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很重要。当A38被无碱基残基或在整个实验pH范围内预期保持相同质子化状态的具有非常高或非常低pK(a)值的核碱基类似物取代时,活性的pH依赖性转变向碱性方向移动了几个pH单位。某些与腺嘌呤的脒基共享的外源核碱基使缺乏A38的无碱基核酶变体恢复了活性。化学拯救反应的pH依赖性也根据拯救核碱基的固有碱性而变化,这进一步证明嘌呤类似物N1位置的质子化状态对拯救活性很重要。这些结果与发夹状核酶催化机制模型一致,在该模型中,与A38的相互作用为过渡态提供静电稳定作用。