Catalytic mechanism of DNA topoisomerase IB
Catalytic mechanism of DNA topoisomerase IB
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DOI：
10.1016/s1097-2765(00)80268-3
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发表时间：
2000-06-01
期刊：
影响因子：
16
通讯作者：
Shuman, S
中科院分区：
文献类型：
--
作者：
Krogh, BO;Shuman, S
IB型拓扑异构酶和酪氨酸重组酶是结构上同源的链转移酶，通过DNA-（3'-磷酸酪氨酸）酶中间体起作用。一组保守的氨基酸（牛痘拓扑异构酶中的Arg-130、Lys-167、Arg-223和His-265）催化酪氨酸酯交换成可剪切的磷酸二酯。我们使用8'-桥接磷硫酸修饰的dna来暗示Lys-167作为一般的酸催化剂。与5’-O相比，5’-S离开组的较低pK(a)恢复了K167A突变体的活性，而对突变体R223A和H265A没有积极的影响。赖氨酸位于一个灵活的发夹环上，它在DNA结合时转移到小槽中。一般酸环构象变化与磷酸基转移共价催化的耦合是拓扑异构酶/重组酶和蛋白磷酸酶超家族共有的几个机制特征之一。
Type IB topoisomerases and tyrosine recombinases are structurally homologous strand transferases that act through DNA-(3'-phosphotyrosyl)-enzyme intermediates. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes transesterification of tyrosine to the scissile phosphodiester. We used 8'-bridging phosphorothiolate-modified DNAs to implicate Lys-167 as a general acid catalyst. The lower pK(a), of the 5'-S leaving group versus 5'-O restored activity to the K167A mutant, whereas there was no positive thio effect for mutants R223A and H265A. The lysine is located atop a flexible hairpin loop, and it shifts into the minor groove upon DNA binding. Coupling of conformational changes in a general acid loop to covalent catalysis of phosphoryl transfer is one of several mechanistic features shared by the topoisomerase/recombinase and protein phosphatase superfamilies.