DNA cleavage by EcoRV endonuclease:: Two metal ions in three metal ion binding sites
DNA cleavage by EcoRV endonuclease:: Two metal ions in three metal ion binding sites
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DOI：
10.1021/bi0499056
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发表时间：
2004-06-08
期刊：
影响因子：
2.9
通讯作者：
Perona, JJ
中科院分区：
文献类型：
--
作者：
Horton, NC;Perona, JJ
EcoRV内切酶突变体K92A和K38A的四种晶体结构为揭示DNA弯曲的机制和金属依赖的磷酸二酯键断裂的结构基础提供了新的视角。在未切割的K92A-DNA-M2+底物复合体中，一个关键的活性部位正电荷的去除导致了一个钠离子在胺氮位置上的结合，这表明该位置的正电荷在切割前稳定DNA急剧弯曲方面起着关键作用。相反，在不同的晶格环境中与DNA和Mn2+离子共结晶的两种K38A结构显示出裂解产物络合物，与所有以前的EcoRV结构相比，具有共同的、新的剪刀状磷酸基团构象。在这些结构中，释放的5‘-磷酸和3’-OH基团保持紧密的并列，并与两个连接保守活性中心羧酸盐的Mn2+离子保持着紧密的并列。剪切型磷酸盐位于野生型酶的活性前底物和活性后产物复合体的中间位置。Mn2+离子占据了先前在预活性络合物中描述的三个位置中的两个，并且似乎被定位为生成亲核氢氧化物离子，以补偿过渡态中初始的额外负电荷，并电离第二水以使3‘-氧阴离子质子化。这些发现与早期的X射线和荧光研究相一致，表明了一种新的机制，即在第三个不同的位置上，单个初始结合的金属离子经历了位置的移动，同时剪刀状的磷酸盐移动到更深的活性位置裂隙中。这重新配置了局部环境，以允许第二金属离子结合，然后向五价过渡态移动。这里提出的新机制体现了以前提出的两金属和三金属催化模型的关键特征，并提供了一种立体化学途径的观点，该途径整合了从许多实验室的详尽研究中获得的大量结构和功能数据。
Four crystal structures of EcoRV endonuclease mutants K92A and K38A provide new insight into the mechanism of DNA bending and the structural basis for metal-dependent phosphodiester bond cleavage. The removal of a key active site positive charge in the uncleaved K92A-DNA-M2+ substrate complex results in binding of a sodium ion in the position of the amine nitrogen, suggesting a key role for a positive charge at this position in stabilizing the sharp DNA bend prior to cleavage. By contrast, two structures of K38A cocrystallized with DNA and Mn2+ ions in different lattice environments reveal cleaved product complexes featuring a common, novel conformation of the scissile phosphate group as compared to all previous EcoRV structures. In these structures, the released 5'-phosphate and 3'-OH groups remain in close juxtaposition with each other and with two Mn2+ ions that bridge the conserved active site carboxylates. The scissile phosphates are found midway between their positions in the prereactive substrate and postreactive product complexes of the wild-type enzyme. Mn2+ ions occupy two of the three sites previously described in the prereactive complexes and are plausibly positioned to generate the nucleophilic hydroxide ion, to compensate for the incipient additional negative charge in the transition state, and to ionize a second water for protonation of the 3'-oxyanion. Reconciliation of these findings with earlier X-ray and fluorescence studies suggests a novel mechanism in which a single initially bound metal ion in a third distinct site undergoes a shift in position together with movement of the scissile phosphate deeper into the active site cleft. This reconfigures the local environment to permit binding of the second metal ion followed by movement toward the pentacovalent transition state. The new mechanism suggested here embodies key features of previously proposed two- and three-metal catalytic models, and offers a view of the stereochemical pathway that integrates much of the copious structural and functional data that are available from exhaustive studies in many laboratories.