Mechanistic Characterization of the HDV Genomic Ribozyme: Solvent Isotope Effects and Proton Inventories in the Absence of Divalent Metal Ions Support C75 as the General Acid
Mechanistic Characterization of the HDV Genomic Ribozyme: Solvent Isotope Effects and Proton Inventories in the Absence of Divalent Metal Ions Support C75 as the General Acid
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DOI：
10.1021/ja801816k
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发表时间：
2008-11-05
影响因子：
15
通讯作者：
Bevilacqua, Philip C.
中科院分区：
文献类型：
--
作者：
Cerrone-Szakal, Andrea L.;Siegfried, Nathan A.;Bevilacqua, Philip C.
丁型肝炎病毒（HDV）核酶利用核碱基C75和水合Mg 2+离子作为在生理盐下磷酸二酯键断裂的一般酸碱催化剂。一个机制框架已被先进，涉及一个Mg 2+独立和两个Mg 2+依赖性通道。野生型（WT）核酶在无Mg 2+通道中的速率-pH曲线相对于完全Mg 2+依赖性通道是反向的，每个通道具有近中性pK（a）。速率-pH曲线的反转被用作C75在存在和不存在Mg 2+的情况下充当一般酸的机械论点的关键。然而，随后对双突变体（DM）核酶的研究表明，在不存在Mg 2+的情况下观察到的WT的pK（a）来自C41（一种结构核碱基）的电离。为了进一步研究这一点，我们在不存在Mg 2+的情况下获得了WT和DM的速率-pH/pD曲线和质子库存。校正了离子强度对氢离子活度和pH计读数的影响。结果由模型调节，其中观察到WT的无Mg 2 + pKa来自C75的电离，DM反应性受到C41质子化的影响。布朗斯台德碱似乎是水或氢氧化物离子，这取决于pH值。观察到的pK（a）与模型寡核苷酸的盐依赖性pH滴定以及静电计算有关，这支持在不存在Mg 2+的情况下C75的局部环境与在存在Mg 2+的情况下相似，并且不受本体离子的影响。因此，C75作为一般酸的催化作用似乎不依赖于二价离子或布朗斯台德碱的特性。
The hepatitis delta virus (HDV) ribozyme uses the nucleobase C75 and a hydrated Mg2+ ion as the general acid-base catalysts in phosphodiester bond cleavage at physiological salt. A mechanistic framework has been advanced that involves one Mg2+-independent and two Mg2+-dependent channels. The rate-pH profile for wild-type (WT) ribozyme in the Mg2+-free channel is inverted relative to the fully Mg2+-dependent channel, with each having a near-neutral pK(a). Inversion of the rate-pH profile was used as the crux of a mechanistic argument that C75 serves as general acid both in the presence and absence of Mg2+. However, subsequent studies on a double mutant (DM) ribozyme suggested that the pK(a) observed for WT in the absence of Mg2+ arises from ionization of C41, a structural nucleobase. To investigate this further, we acquired rate-pH/pD profiles and proton inventories for WT and DM in the absence of Mg2+. Corrections were made for effects of ionic strength on hydrogen ion activity and pH meter readings. Results are accommodated by a model wherein the Mg2+-free pKa observed for WT arises from ionization of C75, and DM reactivity is compromised by protonation of C41. The Bronsted base appears to be water or hydroxide ion depending on pH. The observed pK(a)'s are related to salt-dependent pH titrations of a model oligonucleotide, as well as electrostatic calculations, which support the local environment for C75 in the absence of Mg2+ being similar to that in the presence of Mg2+ and impervious to bulk ions. Accordingly, the catalytic role of C75 as the general acid does not appear to depend on divalent ions or the identity of the Bronsted base.