The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine
The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine
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DOI：
10.1074/jbc.m111.234906
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发表时间：
2011-05-20
影响因子：
4.8
通讯作者：
Fedor, Martha J.
中科院分区：
文献类型：
--
作者：
Cottrell, Joseph W.;Scott, Lincoln G.;Fedor, Martha J.
鉴于人类和细菌基因表达可以通过 RNA 自切割来调节的令人信服的证据，了解自切割核酶如何介导催化作用至关重要。发夹核酶通过不需要二价金属阳离子的机制催化可逆磷酸二酯键裂解。先前的结构和生化证据表明活性位点腺苷 A38 的脒基团参与了 pH 依赖性催化步骤。我们开发了一种基于 8-氮杂腺苷 (8azaA) 的 pH 依赖性荧光来确定活性核酶中微观 pK(a) 值的方法。我们将第 38 位 8azaA 电离的微观 pKa 与第 38 位具有独特 8azaA 的全功能发夹核酶中自裂解反应的表观 pK(a) 进行了比较。微观和表观 pK(a) 值几乎相同，这证明 A38 质子化导致催化活性随 pH 值降低而降低。这些结果表明 A38 的中性非质子化形式处于过渡态，涉及 5'-氧-磷键的形成。
Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pK(a) values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pKa for ionization of 8azaA at position 38 with the apparent pK(a) for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK(a) values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5'-oxygen-phosphorus bond.