Zhou T, Rosen B P
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
J Biol Chem. 1999 May 14;274(20):13854-8. doi: 10.1074/jbc.274.20.13854.
The ATPase activity of ArsA, the catalytic subunit of the plasmid-encoded, ATP-dependent extrusion pump for arsenicals and antimonials in Escherichia coli, is allosterically activated by arsenite or antimonite. Magnesium is essential for ATPase activity. To examine the role of Asp45, mutants were constructed in which Asp45 was changed to Glu, Asn, or Ala. Cells expressing these mutated arsA genes lost arsenite resistance to varying degrees. Purified D45A and D45N enzymes were inactive. The purified D45E enzyme exhibited approximately 5% of the wild type activity with about a 5-fold decrease in affinity for Mg2+. Intrinsic tryptophan fluorescence was used to probe Mg2+ binding. ArsA containing only Trp159 exhibited fluorescence enhancement upon the addition of MgATP, which was absent in D45N and D45A. As another measure of conformation, limited trypsin digestion was used to estimate the surface accessibility of residues in ArsA. ATP and Sb(III) synergistically protected wild type ArsA from trypsin digestion. Subsequent addition of Mg2+ increased trypsin sensitivity. D45N and D45A remained protected by ATP and Sb(III) but lost the Mg2+ effect. D45E exhibited an intermediate Mg2+ response. These results indicate that Asp45 is a Mg2+-responsive residue, consistent with its function as a Mg2+ ligand.
ArsA是大肠杆菌中由质粒编码的、依赖ATP的砷化物和锑化物外排泵的催化亚基，其ATP酶活性受到亚砷酸盐或亚锑酸盐的变构激活。镁对于ATP酶活性至关重要。为了研究Asp45的作用，构建了将Asp45分别替换为Glu、Asn或Ala的突变体。表达这些突变arsA基因的细胞不同程度地丧失了对亚砷酸盐的抗性。纯化的D45A和D45N酶没有活性。纯化的D45E酶表现出约5%的野生型活性，对Mg2+的亲和力下降了约5倍。利用内源色氨酸荧光来探测Mg2+结合。仅含有Trp159的ArsA在添加MgATP后荧光增强，而D45N和D45A则没有这种现象。作为构象的另一种衡量方法，采用有限胰蛋白酶消化来估计ArsA中残基的表面可及性。ATP和Sb(III)协同保护野生型ArsA不被胰蛋白酶消化。随后添加Mg2+会增加对胰蛋白酶的敏感性。D45N和D45A仍受到ATP和Sb(III)的保护，但失去了Mg2+的影响。D45E表现出中等程度的Mg2+反应。这些结果表明Asp45是一个对Mg2+有反应的残基，与其作为Mg2+配体的功能一致。
