Co-crystal of Escherichia coli RNase HI with Mn2+ ions reveals two divalent metals bound in the active site
Co-crystal of Escherichia coli RNase HI with Mn2+ ions reveals two divalent metals bound in the active site
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DOI：
10.1074/jbc.m009626200
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发表时间：
2001-03-09
影响因子：
4.8
通讯作者：
Marqusee, S
中科院分区：
文献类型：
--
作者：
Goedken, ER;Marqusee, S
核糖核酸酶H（RNase H）选择性地降解RNA的RNA链。二价阳离子依赖性的DNA杂交。先前的结构研究揭示了在大肠杆菌RNase HI中的单个Mg 2+离子结合位点。于：人免疫缺陷病毒逆转录酶的相关RNase H结构域的晶体结构，然而，观察到两个Mn 2+离子，表明不同的金属结合模式。大肠杆菌RNase HI在Mg 2+或Mn 2+离子存在下显示催化活性，但是：这两种金属显示出显著不同的最佳浓度。Mg 2+离子需要以毫摩尔浓度存在，但Mn 2+离子仅需要以微摩尔量存在。基于对金属的依赖性。coli RNase HI活性，我们提出了一个激活/衰减模型，其中一种金属是催化所需的，第二种金属的结合是抑制性的。我们现在已经以1.9埃的分辨率解决了大肠杆菌RNase HI与Mn 2+离子的共晶结构。两个八面体配位的Mn 2+离子被认为是绑定到酶的活性位点。残基Asp-10、Glu-48和Asp-70与第一（活化）金属形成直接（内球）配位接触，而残基Asp-10和Asp-134与第二（衰减）金属形成直接接触。这种结构与生物化学证据一致，表明两种金属离子可以结合RNA酶H，但配体第二种离子抑制RNA酶H活性。
Ribonuclease H (RNase H) selectively degrades the RNA strand of RNA . DNA hybrids in a divalent cation-dependent manner. Previous structural studies revealed a single Mg2+ ion-binding site in Escherichia coli RNase HI. In: the crystal structure of the related RNase H domain: of human immunodeficiency virus reverse transcriptase, however, two Mn2+ ions were observed suggesting a different mode of metal binding. E, coli RNase HI shows catalytic activity in the presence of Mg2+ or Mn2+ ions, but: these two metals show strikingly different optimal concentrations. Mg2+ ions are required in millimolar concentrations, but Mn2+ ions are only required-in micromolar quantities. Based upon the metal dependence off. coli RNase HI activity, we proposed an activation/attenuation model in which one metal is required for catalysis, and binding of a second metal is inhibitory. We have now solved the co-crystal structure of E, coli RNase HI with Mn2+ ions at 1.9-Angstrom resolution. Two octahedrally coordinated Mn2+ ions are seen to bind to the enzyme-active site. Residues Asp-10, Glu-48, and Asp-70 make direct (inner sphere) coordination contacts to the first (activating) metal, whereas residues Asp-10 and Asp-134 make direct contacts to the second (attenuating) metal. This structure is consistent with biochemical evidence suggesting that two metal ions may bind RNase H but liganding a second ion inhibits RNase H activity.