Liu J, Rosen B P
Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
J Biol Chem. 1997 Aug 22;272(34):21084-9. doi: 10.1074/jbc.272.34.21084.
ArsC encoded by Escherichia coli plasmid R773 catalyzes the reduction of arsenate to arsenite. The enzymatic reaction requires reduced glutathione and glutaredoxin. In this study a direct association between ArsC and glutaredoxin was demonstrated. An arsC gene with six histidine codons added to the 5' end of the gene was constructed, and the resulting ArsC enyzme was shown to be functional. Interaction of the histidine-tagged ArsC and glutaredoxin was examined by Ni2+ affinity chromatography. The association required the presence of reduced glutathione and either the substrate arsenate or a competitive inhibitor, phosphate or sulfate. A free thiolate on glutathione was not required. A tryptophan residue was introduced into ArsC at the 11th position, immediately adjacent to the active site Cys-12. Trp-11 fluorescence was quenched upon addition of arsenate. Addition of reduced glutathione after arsenate resulted in a rapid increase in fluorescence followed by a slower decay of the signal. These spectroscopic signals were specific for arsenate and reduced glutathione; neither competitive inhibitors nor non-thiol glutathione analogs produced this effect. Cys-12 thiolate was also required. Thus the intrinsic fluorescence of Trp-11 provides a useful probe to investigate the mechanism of this novel reductase.
由大肠杆菌质粒R773编码的ArsC催化砷酸盐还原为亚砷酸盐。该酶促反应需要还原型谷胱甘肽和谷氧还蛋白。在本研究中，证明了ArsC与谷氧还蛋白之间存在直接关联。构建了一个在基因5'端添加了六个组氨酸密码子的arsC基因，结果表明所得的ArsC酶具有功能。通过Ni2+亲和色谱法检测了组氨酸标签化的ArsC与谷氧还蛋白的相互作用。这种关联需要还原型谷胱甘肽以及底物砷酸盐或竞争性抑制剂磷酸盐或硫酸盐的存在。谷胱甘肽上的游离硫醇盐不是必需的。在紧邻活性位点Cys-12的第11位将一个色氨酸残基引入ArsC。加入砷酸盐后，Trp-11荧光猝灭。在砷酸盐之后加入还原型谷胱甘肽导致荧光迅速增加，随后信号缓慢衰减。这些光谱信号对砷酸盐和还原型谷胱甘肽具有特异性；竞争性抑制剂和非硫醇谷胱甘肽类似物均未产生这种效应。Cys-12硫醇盐也是必需的。因此，Trp-11的固有荧光为研究这种新型还原酶的机制提供了一种有用的探针。
