Li J, Rosen B P
Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, MI 48201, USA.
Mol Microbiol. 2000 Jan;35(2):361-7. doi: 10.1046/j.1365-2958.2000.01696.x.
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 N- and 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 5-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 5-residue insertion had the same affinity for ATP and Sb(III) as the wild-type 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.
质粒R773编码一种As(III)/Sb(III)转运ATP酶，该酶赋予大肠杆菌对这些类金属的抗性。该泵的催化亚基ArsA ATP酶由通过25个残基的连接子相连的同源N端和C端核苷酸结合结构域组成。通过缺失5、10、15或23个残基或插入5个甘氨酸残基来研究该连接子序列的作用。表达带有5个残基插入的arsA的细胞具有野生型亚砷酸盐抗性。表达带有缺失的修饰arsA基因的细胞的抗性取决于连接子长度。缺失5或10个残基的细胞表现出抗性略有降低。缺失15或23个残基导致抗性进一步降低。每种改变的ArsA都被纯化。带有5个残基插入的酶对ATP和Sb(III)的亲和力与野生型酶相同。缺失5、10、15或23个残基的酶对Sb(III)和ATP的亲和力均降低。缺失23个残基的酶仅表现出基础ATP酶活性，并且不能被Sb(III)变构激活。这些结果表明，连接子已经进化到一个最佳长度，以便使蛋白质的两半部分彼此正确接触，从而促进催化作用。
