Mg2 +绑定EcoRV内切核酸酶的活性部位:晶体研究配合物与底物和产品DNA 2一项决议。
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Kostrewa D,温克勒颗
Mg2 +绑定EcoRV内切核酸酶的活性部位:晶体研究配合物与底物和产品DNA 2一项决议。
生物化学。1995年1月17日,34 (2):683 - 96。
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7819264 (在PubMed]
- 文摘
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类型II限制性内切核酸酶EcoRV结晶是一个复杂的DNA与衬底undecamer AAAGATATCTT(识别序列强调)。这些晶体衍射更好的解决方案(2)比之前报道的复杂与decamer GGGATATCCC[温克勒,f·K。横幅,d . W。Oefner C。Tsernoglou D。、棕色、r S。、Heathman s P。,布莱恩·r·K。,马丁,p D。Petratos, K。& Wilson, EMBO j . k . s . (1993) 1781 - 1795]。晶体结构包含一个二聚体在不对称单位,解决了复杂分子置换。同样的弯折的DNA构象特征enzyme-bound同源DNA。与Mg2 +水晶,浸泡,显示必要的辅助因子只有一个活性部位的二聚体,和DNA裂解。Mg2 +有一个氧气从易裂开的磷酸二酯组和两个羧酸盐氧,一种形式从Asp90 Asp74和一个八面体配位体的球体。 The scissile phosphodiester group is pulled by 1 A toward the Mg2+. After substrate cleavage in solution, isomorphous crystals containing the enzyme--product--Mg2+ complex were obtained. In this structure, each of the 5'-phosphate groups is bound to two Mg2+. The kinked DNA conformation is essentially maintained, but the two central adenines, 3' to the cleavage sites, form an unusual cross-strand base stacking. The structures have been refined to R factors of 0.16 at 2.1-2.0 A resolution maintaining very good stereochemistry. On the basis of these structures and inspired by recent kinetic data [Vipond, I. B., & Halford, S. E. (1994) Biochemistry (second paper of three in this issue)], we have constructed a transition state model with two metals bound to the scissile phosphorane group.