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首页> 外文期刊>Biochemistry >Model for general acid-base catalysis by the hammerhead ribozyme: pH-activity relationships of G8 and G12 variants at the putative active site
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Model for general acid-base catalysis by the hammerhead ribozyme: pH-activity relationships of G8 and G12 variants at the putative active site

机译：锤头状核酶对一般酸碱催化的模型：假定的活性位点上G8和G12变体的pH活性关系

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摘要

We have used nucleobase substitution and kinetic analysis to test the hypothesis that hammerhead catalysis occurs by a general acid-base mechanism, in which nucleobases are directly involved in deprotonation of the attacking 2'-hydroxyl group and protonation of the 5'-oxygen that serves as the leaving group in the cleavage reaction. We demonstrate that simultaneous substitution of two important nucleobases, G8 and G12, with 2,6-diaminopurine shifts the pH optimum of the cleavage reaction from greater than 9.5 to approximately 6.8 in two different hammerhead constructs. Controls involving substitution with other nucleobases and combinations of nucleobases at G5, G8, and/or G12 do not show this behavior. The observed changes in the pH-rate behavior are consistent with a mechanism in which NI protonation-deprotonation events of guanine or 2,6-diaminopurine at positions 8 and 12 are essential for catalysis. Further support for the participation of G8 and G12 comes from photochemical cross-linking experiments, which show that G8 and G12 can stack upon the two substrate nucleobases at the reactive linkage, G(or U)1.1 and C17 (Heckman, J. E., Lambert, D., and Burke, J. A (2005) Photocrosslinking detects a compact active structure of the hammerhead ribozyme, Biochemistry 44, 41484156). Together, these results support a model in which the hammerhead undergoes a transient conformational change into a catalytically active structure, in which stacking of G8 and G12 upon the nucleobases spanning the cleavage site provides an appropriate architecture for general acid-base catalysis. The hammerhead and hairpin ribozymes may share similarities in the organization of their active sites and their catalytic mechanism.
机译：我们已经使用核碱基取代和动力学分析来检验假说，锤头催化是通过一般的酸碱机制发生的，其中核碱基直接参与攻击的2'-羟基的去质子化和提供服务的5'-氧的质子化作为裂解反应中的离去基团。我们证明，同时用2，6-二氨基嘌呤取代两个重要的核碱基G8和G12，可在两个不同的锤头构建物中将裂解反应的最适pH从大于9.5移至约6.8。涉及用其他核碱基取代和G5，G8和/或G12处的核碱基组合的对照未显示此行为。观察到的pH速率变化与以下机理一致：在第8位和第12位的鸟嘌呤或2,6-二氨基嘌呤的NI质子化-去质子化事件对于催化至关重要。光化学交联实验进一步支持G8和G12的参与，该实验表明G8和G12可以在反应性键G（或U）1.1和C17上堆叠在两个底物核碱基上（Heckman，JE，Lambert， D.和Burke，J.A（2005），光交联检测锤头状核酶的致密活性结构，Biochemistry 44，41484156）。在一起，这些结果支持了一个模型，其中锤头经历了短暂的构象变化，变成了催化活性结构，其中G8和G12在跨越裂解位点的核碱基上的堆叠提供了适用于一般酸碱催化的结构。锤头和发夹状核酶在活性位点的组织和催化机理上可能有相似之处。

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