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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage
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Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage

机译：胞嘧啶侧链参与质子转移在核酶自我切割的速率确定步骤中

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摘要

Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reac- tion. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining Step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76△) ribozymes in D_2O and H_2O. In both reactons, a similar kinetic isotope effed and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction sup- ported a mechanism of a single proton transfer at the transition State. this proton transfer Step was further characterized by exogenous base rescue of a C76A mutant with Cytosine and imidazole analogues. For the imidazole analogues that rescued activity the apparent pKa of the rescue reaction, measured under k_cat/KM conditions, correlated with the pKa of the base. From these data a Bronsted coefficient (P) of 0.51 was determined for the base-rescued reaction of C76A. This value is consistent with that expeded for proton transfer in the transition State. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage.
机译：有人提出，肝炎三角洲病毒的核酶可以在自我切割反应中使用活性位胞嘧啶作为酸碱催化剂。在这项研究中，我们用反基因组核酶更详细地研究了胞嘧啶的作用。速率确定步骤中质子转移涉及胞嘧啶76（C76）的证据是通过检查D_2O和H_2O中野生型和咪唑缓冲液拯救的C76（C76△）核酶的裂解活性而获得的。在两个反应器中，相似的动力学同位素都起作用并且表观pKa发生变化，表明缓冲剂在功能上替代了质子转移中的侧链。野生型反应的质子库存支持在过渡状态下单个质子转移的机制。该质子转移步骤的进一步特征在于用胞嘧啶和咪唑类似物对C76A突变体进行外源碱基拯救。对于挽救活性的咪唑类似物，在k_cat / KM条件下测得的挽救反应的表观pKa与碱基的pKa相关。根据这些数据，对于碱拯救的C76A反应，得出的布朗斯台德系数（P）为0.51。该值与过渡态中质子转移的预期值一致。总之，这些数据为RNA侧链直接参与野生型核酶的普通酸或普通碱基催化以促进RNA裂解的机制提供了有力的支持。

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