pKa perturbation in genomic hepatitis delta virus ribozyme catalysis evidenced by nucleotide analogue interference mapping
pKa perturbation in genomic hepatitis delta virus ribozyme catalysis evidenced by nucleotide analogue interference mapping
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DOI：
10.1021/bi011816v
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发表时间：
2002-03-19
期刊：
影响因子：
2.9
通讯作者：
Strobel, SA
中科院分区：
文献类型：
--
作者：
Oyelere, AK;Kardon, JR;Strobel, SA
丁型肝炎病毒（HDV）核酶是第一个提出使用质子转移机制进行催化的RNA酶。先前的生物化学证据表明，基因组HDV核酶使用胞嘧啶75促进顺式切割，胞嘧啶75的pK（a）在活性位点内被扰动。在这里，我们提出了进一步的生化证据参与质子转移的C75，以及证据支持一个合理的机制C75 pKa扰动。用具有改变的N3 pK（a）s的C类似物进行的核苷酸类似物干扰作图（NAIM）实验证明了C75电离在HDV顺式切割反应中的重要性。具有增强的N3酸度的C类似物的pH依赖性干扰拯救表明C75是必须被质子化以获得核酶活性的唯一胞苷残基。此外，干扰分析与假异胞苷，一个电荷中性模拟的C与质子化的N3，显示出与质子转移一致的模式，可能是从C75 N3的5 '-氧阴离子离去基团在顺式裂解反应。强烈的pH值无关的抑制核酶功能也发生在C75与C类似物，缺乏N4氨基，暗示环外胺的关键相互作用的活性位点。与氨基的相互作用可能在扰乱C75 N3 pK（a）中发挥重要作用。质子化的C41已被提出是重要的核酶活性，但是，没有干扰C41观察到在这个类似物系列，反对C41质子化的功能作用。这些数据支持一种模型，其中基因组HDV核酶的C75在其顺式切割反应期间充当一般酸，并提供了RNA如何的一瞥。以类似于蛋白酶的方式，可能利用局部环境电子调制来催化反应。
The Hepatitis Delta Virus (HDV) ribozyme was the first RNA enzyme proposed to use a proton-transfer mechanism for catalysis. Previous biochemical evidence suggested that the genomic HDV ribozyme promotes cis-cleavage using cytosine 75 whose pK(a) is perturbed within the active site. Here we present further biochemical evidence for the involvement of C75 in proton transfer, as well as evidence to support a plausible mechanism for C75 pKa perturbation. Nucleotide analogue interference mapping (NAIM) experiments with C analogues having altered N3 pK(a)s demonstrate the importance of C75 ionization in the HDV cis-cleavage reaction. pH-dependent interference rescue with C analogues having enhanced N3 acidity indicates that C75 is the only cytidine residue that must be protonated for ribozyme activity. Furthermore, interference analysis with pseudoisocytidine, a charge-neutral mimic of a C with a protonated N3, shows a pattern consistent with proton transfer, possibly from the C75 N3 to the 5'-oxyanion leaving group during the cis-cleavage reaction. Strong pH-independent inhibition of ribozyme function also occurs at C75 with a C analogue that lacks the N4 amino group, implicating the exocyclic amine in critical interactions in the active site. Interactions with the amino group may play an important role in perturbing the C75 N3 pK(a). Protonation of C41 has been proposed to be important for ribozyme activity; however, no interference at C41 was observed in this analogue series, which argues against a functional role for C41 protonation. These data support a model wherein C75 of the genomic HDV ribozyme acts as a general acid during its cis-cleavage reaction, and provide a glimpse into how RNAs. in a manner similar to protein enzymes, might employ local environmental electronic modulation to catalyze reactions.