Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme
Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme
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DOI：
10.1073/pnas.1004255107
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发表时间：
2010-06-29
影响因子：
11.1
通讯作者：
Lilley, David M. J.
中科院分区：
文献类型：
--
作者：
Wilson, Timothy J.;Li, Nan-Sheng;Lilley, David M. J.
现有的证据表明，Varkud卫星（VS）核酶加速使用一般的酸碱催化的特定磷酸二酯键的切割。关键功能是核酶的螺旋VI中的腺嘌呤756的核碱基和底物茎环中的鸟嘌呤638。这导致反应速率对pH的钟形依赖性，对应于pK（a）= 5.2和8.4的组。然而，从这些数据中不可能确定哪个核碱基是酸，哪个是碱。因此，我们已经制备了基质，其中易断裂的磷酸盐的5'氧被硫取代。这使离去基团不稳定，从而消除了对一般酸催化的需要。这种取代恢复了高度受损的A756 G核酶的全部活性，与未修饰的核酶中A756的一般酸催化一致。硫代磷酸酯修饰的底物的裂解的pH依赖性与通过位置638处的核碱基的一般碱催化一致。我们的结论是底物的VS核酶的切割是由G638的2 '-O亲核试剂的去质子化和A756的5'-O离去基团的质子化催化的。
Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pK(a) = 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5' oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolate-modified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2'-O nucleophile by G638 and protonation of the 5'-O leaving group by A756.