Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage
Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage
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DOI：
10.1073/pnas.98.4.1489
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发表时间：
2001-02-13
影响因子：
11.1
通讯作者：
Been, MD
中科院分区：
文献类型：
--
作者：
Shih, I;Been, MD
丁型肝炎病毒的核酶已被提出在自裂解反应中使用活性位点胞嘧啶作为酸碱催化剂。在这项研究中，我们更详细地研究了胞嘧啶与抗基因组核酶的作用，通过检测野生型和咪唑缓冲修复的C76-缺失（C76 Delta）核酶在D2O和H2O中的裂解活性，得到了质子转移在速率决定步骤中涉及胞嘧啶76 （C76）的证据。在这两个反应中，相似的动力学同位素效应和表观pKa的移动表明缓冲液在质子转移中功能性地取代了侧链，野生型反应的质子库存支持过渡态的单质子转移机制。通过胞嘧啶和咪唑类似物对C76 δ突变体的外源碱基修复，进一步表征了这一质子转移步骤。对于拯救活性的咪唑类似物，在k(cat)/ k - m条件下测量的拯救反应的表观pKa与碱的pKa相关。根据这些数据，确定了C76 δ的碱拯救反应的Bronsted系数（beta）为0.51，该值与过渡态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一般酸或一般碱催化以促进RNA切割的机制提供了强有力的支持。
Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. 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 Delta) ribozymes in D2O and H2O. In both reactions, a similar kinetic isotope effect 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 supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76 Delta mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under k(cat)/K-M conditions, correlated with the pKa of the base. From these data a Bronsted coefficient (beta) of 0.51 was determined for the base-rescued reaction of C76 Delta This value is consistent with that expected 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.