Liu J, Gladysheva T B, Lee L, Rosen B P
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Biochemistry. 1995 Oct 17;34(41):13472-6. doi: 10.1021/bi00041a026.
The ArsC protein encoded by the arsenical resistance operon of plasmid R773 catalyzes the reduction of arsenate to arsenite in Escherichia coli. The reductase has been shown to require glutathione and glutaredoxin, suggesting that thiol chemistry might be involved in the reaction mechanism. The ArsC arsenate reductase has two cysteinyl residues, Cys12 and Cys106. By a combination of random and site-specific mutagenesis, Cys12 was altered to four other amino acid residues. Cells expressing any of those arsC genes were sensitive to arsenate. The ArsCC12S protein was purified and found to be catalytically inactive. Cys106 was altered separately to seryl, glycyl, and valyl residues. Cells expressing arsCC106S, arsCC106G, and arsCC106V genes retained arsenate resistance, and the purified C106S and C106G proteins had reductase activity. Both wild-type ArsC and C106S proteins were inactivated by iodoacetate. In the native enzyme only Cys12 was alkylate by iodoacetate; Cys106 was alkylated only if the enzyme was first denatured. In the presence of the substrate, arsenate, or competitive inhibitors, phosphate or sulfate, the rate of alkylation was reduced. Reductase activity was inhibited by N-ethylmaleimide and could be protected by arsenate. These results suggest Cys12 is an active-site residue essential for catalysis by the arsenate reductase.
质粒R773的抗砷操纵子编码的ArsC蛋白在大肠杆菌中催化砷酸盐还原为亚砷酸盐。已证明该还原酶需要谷胱甘肽和谷氧还蛋白，这表明硫醇化学可能参与了反应机制。ArsC砷酸盐还原酶有两个半胱氨酸残基，即Cys12和Cys106。通过随机诱变和定点诱变相结合的方法，将Cys12替换为其他四个氨基酸残基。表达这些arsC基因中任何一个的细胞对砷酸盐敏感。纯化的ArsCC12S蛋白没有催化活性。分别将Cys106替换为丝氨酸、甘氨酸和缬氨酸残基。表达arsCC106S、arsCC106G和arsCC106V基因的细胞仍具有抗砷酸盐能力，纯化的C106S和C106G蛋白具有还原酶活性。野生型ArsC和C106S蛋白都被碘乙酸灭活。在天然酶中，只有Cys12被碘乙酸烷基化；只有当酶首先变性时，Cys106才会被烷基化。在底物砷酸盐或竞争性抑制剂磷酸盐或硫酸盐存在的情况下，烷基化速率降低。还原酶活性受到N-乙基马来酰亚胺的抑制，并且可以被砷酸盐保护。这些结果表明Cys12是砷酸盐还原酶催化所必需的活性位点残基。
