Direct pKa measurement of the active-site cytosine in a genomic hepatitis delta virus ribozyme
Direct pKa measurement of the active-site cytosine in a genomic hepatitis delta virus ribozyme
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DOI：
10.1021/ja016091x
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发表时间：
2001-09-05
影响因子：
15
通讯作者：
Doudna, JA
中科院分区：
文献类型：
--
作者：
Lupták, A;Ferré-D'Amaré, AR;Doudna, JA
丁型肝炎病毒核酶已经被提出通过涉及活性位点胞嘧啶的一般酸/碱机制进行自切割，基于来自切割产物的晶体结构和动力学测量的证据。为了确定基因组核酶中的胞嘧啶（C75）是否具有与其作为一般酸或碱的作用一致的改变的pK（a），我们使用C-13 NMR来确定其在核酶产物形式中的微观pK（a）。测得的pK（a）与游离核苷或碱基配对胞嘧啶的pK（a）有适度偏移，并且与动力学测得的表观反应pK（a）具有相同的二价金属离子依赖性。然而，在测试的所有条件下，微观pK（a）低于表观反应pK（a），支持C75在生理pH下以核酶产物形式去质子化的模型。虽然其他结果表明pK（a）在核酶的反应物状态下没有变化，这些数据不能排除在酯交换反应的中间状态中C75 pK（a）的升高。
Hepatitis delta virus ribozymes have been proposed to perform self-cleavage via a general acid/base mechanism involving an active-site cytosine, based on evidence from both a crystal structure of the cleavage product and kinetic measurements. To determine whether this cytosine (C75) in the genomic ribozyme has an altered pK(a) consistent with its role as a general acid or base, we used C-13 NMR to determine its microscopic pK(a) in the product form of the ribozyme. The measured pK(a) is moderately shifted from that of a free nucleoside or a base-paired cytosine and has the same divalent metal ion dependence as the apparent reaction pK(a)'s measured kinetically. However, under all conditions tested, the microscopic pK(a) is lower than the apparent reaction pK(a), supporting a model in which C75 is deprotonated in the product form of the ribozyme at physiological pH. While additional results suggest that the pK(a) is not shifted in the reactant state of the ribozyme, these data cannot rule out elevation of the C75 pK(a) in an intermediate state of the transesterification reaction.