The linker peptide of the ArsA ATPase
The linker peptide of the ArsA ATPase
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DOI：
10.1046/j.1365-2958.2000.01696.x
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发表时间：
2000-01-01
影响因子：
3.6
通讯作者：
Rosen, BP
中科院分区：
文献类型：
--
作者：
Li, JX;Rosen, BP
质粒 R773 编码 As(III)/Sb(III) 转位 ATP 酶，赋予大肠杆菌对这些类金属的抗性。泵的催化亚基 ArsA ATP 酶由通过 25 个残基接头连接的同源 N 和 C 端核苷酸结合域组成。通过删除 5、10、15 或 23 个残基或插入 5 个甘氨酸残基来检查该接头序列的作用。表达带有 B 残基插入的 arsA 的细胞具有野生型亚砷酸盐抗性。表达具有缺失的修饰的arsA基因的细胞的抗性取决于接头长度。删除 5 个或 10 个残基的细胞表现出轻微降低的抵抗力。 15或23个残基的缺失导致抗性进一步降低。每个改变的 ArsA 均被纯化。插入 B 残基的酶对 ATP 和 Sb(III) 具有与野生型酶相同的亲和力。具有 5、10、15 或 23 个残基缺失的酶对 Sb(III) 和 ATP 的亲和力均降低。具有 23 个残基缺失的酶仅表现出基础 ATP 酶活性，并且无法被 Sb(III) 变构激活。这些结果表明，接头已经进化到最佳长度，可以使蛋白质的两半彼此适当接触，从而促进催化。
Plasmid R773 encodes an As(III)/Sb(III)-translocating ATPase that confers resistance to those metalloids in Escherichia coli. The catalytic subunit of the pump, the ArsA ATPase, consists of homologous Nand C-terminal nucleotide-binding domains connected by a 25-residue linker. The role of this linker sequence was examined by deletion of five, 10, 15 or 23 residues or insertion of five glycine residues. Cells expressing arsA with the B-residue insertion had wild-type arsenite resistance. Resistance of cells expressing modified arsA genes with deletions was dependent on the linker length. Cells with five or 10 deleted residues exhibited slightly reduced resistance. Deletion of 15 or 23 residues resulted in further decreases in resistance. Each altered ArsA was purified. The enzyme with the B-residue insertion had the same affinity for ATP and Sb(III) as the wildtype enzyme. Enzymes with 5-, 10-, 15- or 23-residue deletions exhibited decreased affinity for both Sb(III) and ATP. The enzyme with a 23-residue deletion exhibited only basal ATPase activity and was unable to be allosterically activated by Sb(III). These results suggest that the linker has evolved to a length optimal for bringing the two halves of the protein into proper contact with each other, facilitating catalysis.