Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis.
Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis.
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DOI：
10.1093/nar/gki197
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发表时间：
2005
影响因子：
14.9
通讯作者：
Nicholson AW
中科院分区：
文献类型：
--
作者：
Sun W;Pertzev A;Nicholson AW
大肠杆菌核糖核酸酶III（RNase III; EC 3.1.24）是一种双链（ds）-RNA特异性核酸内切酶，在多种RNA成熟和衰变途径中发挥关键作用。大肠杆菌RNase III是结构上不同的超家族的成员，该超家族包括Dicer，Dicer是RNA干扰机制中的中心酶。大肠杆菌RNA酶III需要二价金属离子来发挥活性，其中Mg 2+是优选的种类。然而，参与催化作用的金属离子的功能和数量都是未知的。为了获得有关金属离子参与催化的信息，将模型底物R1.1 RNA的裂解速率确定为Mg 2+浓度的函数。应用单周转条件，其中磷酸二酯裂解是限速事件。测得的希尔系数（nH）为2.0 ± 0.1，表明磷酸二酯水解中涉及两个Mg 2+离子。研究还表明，2-羟基-4H-异喹啉-1，3-二酮-一种在其催化位点使用两个二价金属离子的核糖核酸酶抑制剂-抑制大肠杆菌RNase III切割R1.1 RNA。对于Mg 2+负载的反应，化合物的IC 50为14 μM，对于Mn 2+负载的反应，化合物的IC 50为8 μM。该化合物表现出非竞争性抑制动力学，表明它不干扰底物结合。无论是O-甲基化的版本的化合物，也没有未取代的酰亚胺抑制底物裂解，这是一个特定的相互作用的N-羟基酰亚胺与两个紧密定位的二价金属离子一致。一个初步的模型，提出了两个二价金属离子在RNase III催化机制的功能作用。
Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded(ds)-RNA-specific endonuclease with key roles in diverse RNA maturation and decay pathways. E.coli RNase III is a member of a structurally distinct superfamily that includes Dicer, a central enzyme in the mechanism of RNA interference. E.coli RNase III requires a divalent metal ion for activity, with Mg2+ as the preferred species. However, neither the function(s) nor the number of metal ions involved in catalysis is known. To gain information on metal ion involvement in catalysis, the rate of cleavage of the model substrate R1.1 RNA was determined as a function of Mg2+ concentration. Single-turnover conditions were applied, wherein phosphodiester cleavage was the rate-limiting event. The measured Hill coefficient (nH) is 2.0 ± 0.1, indicative of the involvement of two Mg2+ ions in phosphodiester hydrolysis. It is also shown that 2-hydroxy-4H-isoquinoline-1,3-dione—an inhibitor of ribonucleases that employ two divalent metal ions in their catalytic sites—inhibits E.coli RNase III cleavage of R1.1 RNA. The IC50 for the compound is 14 μM for the Mg2+-supported reaction, and 8 μM for the Mn2+-supported reaction. The compound exhibits noncompetitive inhibitory kinetics, indicating that it does not perturb substrate binding. Neither the O-methylated version of the compound nor the unsubstituted imide inhibit substrate cleavage, which is consistent with a specific interaction of the N-hydroxyimide with two closely positioned divalent metal ions. A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism.
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