Mutagenesis and modeling of the peroxiredoxin (Prx) complex with the NMR structure of ATP-bound human sulfiredoxin implicate aspartate 187 of Prx I as the catalytic residue in ATP hydrolysis
Mutagenesis and modeling of the peroxiredoxin (Prx) complex with the NMR structure of ATP-bound human sulfiredoxin implicate aspartate 187 of Prx I as the catalytic residue in ATP hydrolysis
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DOI：
10.1021/bi061824h
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发表时间：
2006-12-26
期刊：
影响因子：
2.9
通讯作者：
Gruschus, James M.
中科院分区：
文献类型：
--
作者：
Lee, Duck-Yeon;Park, Sung Jun;Gruschus, James M.
过氧化物氧还蛋白（Prx）家族某些成员的催化半胱氨酸在过氧化物还原过程中可被超氧化为半胱氨酸亚磺酸。硫氧还蛋白负责过氧化Prx的半胱氨酸亚磺酸（SO2 H）的ATP依赖性还原。在这里，我们报告的NMR溶液结构的人sulfiredoxin（hSrx），无论有和没有绑定ATP，我们模型的复杂的ATP结合hSrx与Prx。结合ATP引起的NMR结构的hSrx只有很小的变化，结合ATP的构象是非常相似的，看到以前报道的ADP-hSrx复合物的X-射线结构。虽然hSrx结合ATP，但它本身不催化水解，并且没有大多数ATP酶和激酶家族蛋白质典型的催化酸残基。为了模拟复合物，使用CHARMM的EMAP将ATP结合的hSrx对接到过氧化的Prx II。在模型复合物中，Prx II的Asn 186（Prx I的Asp 187）与hSrx结合的ATP β-和γ-磷酸基团接触。将Prx I的Asp 187突变为丙氨酸和天冬酰胺，并将突变体与hSrx的结合和活性与野生型进行比较。对于D187 N突变体，结合和水解和还原活性与野生型的那些相当，而对于D187 A，结合未受损，但ATP水解和还原没有发生。建模和诱变分析强烈暗示Asp 187的Prx我作为催化残基负责ATP水解的半胱氨酸亚磺酸还原Prx的hSrx。
The catalytic cysteine of certain members of the peroxiredoxin (Prx) family can be hyperoxidized to cysteinesulfinic acid during reduction of peroxides. Sulfiredoxin is responsible for the ATP-dependent reduction of cysteinesulfinic acid (SO2H) of hyperoxidized Prx. Here we report the NMR solution structure of human sulfiredoxin (hSrx), both with and without bound ATP, and we model the complex of ATP-bound hSrx with Prx. Binding ATP causes only small changes in the NMR structure of hSrx, and the bound ATP conformation is quite similar to that seen for the previously reported X-ray structure of the ADP-hSrx complex. Although hSrx binds ATP, it does not catalyze hydrolysis by itself and has no catalytic acid residue typical of most ATPase and kinase family proteins. For modeling the complex, the ATP-bound hSrx was docked to hyperoxidized Prx II using EMAP of CHARMM. In the model complex, Asn186 of Prx II (Asp187 of Prx I) is in contact with the hSrx-bound ATP beta- and gamma-phosphate groups. Asp187 of Prx I was mutated to alanine and asparagine, and binding and activity of the mutants with hSrx were compared to those of the wild type. For the D187N mutant, both binding and hydrolysis and reduction activities were comparable to those of the wild type, whereas for D187A, binding was unimpaired but ATP hydrolysis and reduction did not occur. The modeling and mutagenesis analyses strongly implicate Asp187 of Prx I as the catalytic residue responsible for ATP hydrolysis in the cysteinesulfinic acid reduction of Prx by hSrx.