His-8 lowers the pK(a) of the essential Cys-12 residue of the ArsC arsenate reductase of plasmid R773
His-8 lowers the pK(a) of the essential Cys-12 residue of the ArsC arsenate reductase of plasmid R773
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DOI：
10.1074/jbc.271.52.33256
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发表时间：
1996-12-27
影响因子：
4.8
通讯作者：
Rosen, BP
中科院分区：
文献类型：
--
作者：
Gladysheva, T;Liu, JY;Rosen, BP
质粒 R773 的 141 个残基 ArsC 砷酸还原酶具有一个必需的半胱氨酸残基 Cys-12。 Cys-12 的 pK(a) 测定为 6.4，而游离半胱氨酸的 pK(a) 为 8.3。研究了 Cys-12 和碱性残基之间形成离子对的可能性。组氨酸修饰试剂焦碳酸二乙酯迅速灭活酶活性。 ArsC 中两个组氨酸残基的密码子 His-8 和 His-88 通过定点诱变进行了改变。表达arsC(H88R)、arsC(H88S)、arsC(H88W)或arsC(H88V)基因的细胞保留了砷酸盐抗性，并且纯化的蛋白质具有野生型水平的还原酶活性。表达arSC(H8P)、arsC(H8S)、arsC(H8G)或arsC(H8R)基因的细胞各自对砷酸盐敏感，并且 纯化的 H8P、H8G 和 H8R 蛋白均缺乏酶活性。使用单一组氨酸蛋白表明，两种组氨酸均与焦碳酸二乙酯反应，但仅与 His-8 反应导致失活。 Cys-12 的 pK(a) 值在 H8R 酶中测定为 6.3，在 H8G 酶中测定为 8.3。这些结果表明 His-8 对于催化活性至关重要，并且第 8 位需要带正电的残基来降低第 12 位半胱氨酸硫醇盐的 pK(a)。
The 141-residue ArsC arsenate reductase of plasmid R773 has an essential cysteine residue, Cys-12. The pK(a) of Cys-12 was determined to be 6.4, compared with a pK(a) of 8.3 for free cysteine. The possibility of the formation of an ion pair between Cys-12 and a basic residue was investigated. Enzymatic activity was rapidly inactivated by the histidine-modifying reagent diethylpyrocarbonate. The codons for the two histidine residues in ArsC, His-8 and His-88, were changed by site directed mutagenesis. Cells expressing arsC(H88R), arsC(H88S), arsC(H88W), or arsC(H88V) genes retained arsenate resistance, and the purified proteins had wild type level of reductase activity, Cells expressing arSC(H8P), arsC(H8S), arsC(H8G), or arsC(H8R), genes were each sensitive to arsenate, and the purified H8P, H8G, and H8R proteins each lacked enzymatic activity, Using the single histidine proteins it was shown that both histidines react with dethylpyrocarbonate but that only reaction with His-8 resulted in inactivation. The pK(a) value of Cys-12 was determined to be 6.3 in the H8R enzyme and 8.3 in the H8G enzyme. These results indicate that His-8 is essential for catalytic activity and that a positively charged residue is required at position 8 to lower the pK(a) of the cysteine thiolate at position 12.