Inhibition of EcoRV endonuclease by deoxyribo-3′-S-phosphorothiolates:: A high-resolution X-ray crystallographic study
Inhibition of EcoRV endonuclease by deoxyribo-3′-S-phosphorothiolates:: A high-resolution X-ray crystallographic study
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DOI：
10.1021/ja993719j
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发表时间：
2000-04-12
影响因子：
15
通讯作者：
Perona, JJ
中科院分区：
文献类型：
--
作者：
Horton, NC;Connolly, BA;Perona, JJ
限制性内切核酸酶EcoRV与双链DNA底物类似物结合，在两个易断裂的磷酸盐处具有脱氧核糖-3 '-S-硫代磷酸酯键，这三种限制性内切核酸酶EcoRV的高分辨率结构被提出。在这些结构中的每一个与Mg 2+，Mn 2+，或Ca 2+离子共结晶，非酯化pro-S氧的易裂磷酸盐不再直接连接二价阳离子，如观察到的未修饰的复合物。相反，所有三种结构中的一种金属离子向DNA的相邻3 '-磷酸转移，占据与先前在EroRV T93 A/DNA/Ca 2+复合物中观察到的几乎相同的位置（N. C. Horton等人Proc. Natl.美国科学院U.S.A. 1998，95，13489）。每个结构中的第二个二价金属离子桥接Asp 74和Glu 45（74/45位点）的羧酸根基团，如在野生型和T93 A共晶体中也看到的。这些复合物中未裂解的3 '-S-硫代磷酸酯DNA与未修饰的双链体的构象相比仅略微变形。动力学测量表明，对于活性金属Mg 2+、Mn 2+和Co 2+中的每一种，用于类似物裂解的化学步骤的速率严重降低，并且亲硫Mn 2+、Cd 2+和Zn 2+阳离子不提供可测量的活性重建。亲硫金属不能提高活性与来自先前晶体结构的催化模型一致，这表明金属离子与3 '-氧的连接是通过内球水分子介导的，而不是通过直接相互作用。这些结构表明，3 '-S-硫代磷酸酯类似物抗切割，因为桥接硫排除了二价金属离子与易切割的磷酸盐上的任何位置的内球连接。这将EcoRV中的抑制机制与在DNA聚合酶I的3 '-5'外切核酸酶活性位点中起作用的抑制机制区分开来（C. A. Brautigam等人，Biochemistry，1999，38，696），并且也可能来自其它酶，所述其它酶也通过直接金属连接催化磷酰基转移至3 '-氧离去基团。
Three high-resolution structures of the restriction endonuclease EcoRV bound to a duplex DNA substrate analogue with deoxyribo-3'-S-phosphorothiolate linkages at both scissile phosphates are presented. In each of these structures cocrystallized with Mg2+, Mn2+, or Ca2+ ions, the nonesterified pro-S oxygen of the scissile phosphate no longer directly ligates a divalent cation, as is observed for the unmodified complex. Instead, one metal ion in all three structures is shifted toward the adjacent 3'-phosphate of the DNA, to occupy a position nearly identical to that previously observed in an EroRV T93A/DNA/Ca2+ complex (N. C. Horton et al.. Proc. Natl. Acad Sci. U.S.A. 1998, 95, 13489). A second divalent metal ion in each structure bridges the carboxylate groups of Asp74 and Glu45 (74/45 site), as also seen in both wild-type and T93A cocrystals. The uncleaved 3'-S-phosphorothiolate DNAs in these complexes are only slightly distorted from the conformation of the unmodified duplex. Kinetic measurements show that the rate of the chemical step For analogue cleavage is severely reduced for each of the active metals Mg2+, Mn2+, and Co2+, and that the thiophilic Mn2+, Cd2+, and Zn2+ cations do not provide a measurable reconstitution of activity. The inability of thiophilic metals to improve activity is consistent with models for catalysis derived from previous crystal structures, which indicate that ligation of a metal ion to the 3'-oxygen is mediated through an inner-sphere water molecule rather than by direct interaction. The structures suggest that 3'-S-phosphorothiolale analogues resist cleavage because the bridging sulfur excludes inner-sphere ligation of divalent metal ions to any position on the scissile phosphate. This distinguishes the inhibitory mechanism in EcoRV from that operative in the 3'-5' exonuclease active site of DNA polymerase I (C. A. Brautigam et al., Biochemistry, 1999, 38, 696), and likely as well from other enzymes which also catalyze phosphoryl transfer via direct metal ligation to the 3'-oxygen leaving group.