Chemical rescue, multiple ionizable groups, and general acid-base catalysis in the HDV genomic ribozyme
Chemical rescue, multiple ionizable groups, and general acid-base catalysis in the HDV genomic ribozyme
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DOI：
10.1261/rna.14106
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发表时间：
2006-07-01
期刊：
影响因子：
4.5
通讯作者：
Been, Michael D.
中科院分区：
文献类型：
--
作者：
Perrotta, Anne T.;Wadkins, Timothy S.;Been, Michael D.
在来自丁型肝炎病毒（HDV）基因组链RNA的核酶中，提出胞嘧啶侧链以促进反应过渡态中的质子转移，并且因此充当一般的酸碱催化剂。这种活性位点胞嘧啶（C75）的突变使RNA切割速率降低了100万倍，但加入外源胞嘧啶和某些核碱基或咪唑类似物可以部分挽救这些突变体的活性。然而，拯救反应的pH-速率曲线是钟形的，并且只有一条腿的pH-速率曲线可以归因于外源性核碱基或缓冲液的电离。当第二个潜在的可电离的核碱基（C41）被去除时，钟形曲线的一条腿在化学拯救反应中被消除。利用该结构，从与缓冲液的溶液pKa相关的pH-速率曲线确定的表观pK（a），以及缓冲液对速率增强的贡献可以在自由能或布朗斯台德图中直接评估。缓冲液的酸解离常数与裂解速率常数（布朗斯台德值，β，=类似于0.5）之间的自由能关系与缓冲液充当一般酸碱催化剂的机制一致。这些数据支持的假设，胞嘧啶75，在完整的核酶，作为一个通用的酸碱催化剂。
In the ribozyme from the hepatitis delta virus (HDV) genomic strand RNA, a cytosine side chain is proposed to facilitate proton transfer in the transition state of the reaction and, thus, act as a general acid-base catalyst. Mutation of this active-site cytosine (C75) reduced RNA cleavage rates by as much as one million-fold, but addition of exogenous cytosine and certain nucleobase or imidazole analogs can partially rescue activity in these mutants. However, pH-rate profiles for the rescued reactions were bell shaped, and only one leg of the pH-rate curve could be attributed to ionization of the exogenous nucleobase or buffer. When a second potential ionizable nucleobase (C41) was removed, one leg of the bell-shaped curve was eliminated in the chemical-rescue reaction. With this construct, the apparent pK(a) determined from the pH-rate profile correlated with the solution pKa of the buffer, and the contribution of the buffer to the rate enhancement could be directly evaluated in a free-energy or Bronsted plot. The free-energy relationship between the acid dissociation constant of the buffer and the rate constant for cleavage (Bronsted value, beta, = similar to 0.5) was consistent with a mechanism in which the buffer acted as a general acid-base catalyst. These data support the hypothesis that cytosine 75, in the intact ribozyme, acts as a general acid-base catalyst.