Buurman E T, Kim K T, Epstein W
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.
J Biol Chem. 1995 Mar 24;270(12):6678-85. doi: 10.1074/jbc.270.12.6678.
Substrate binding sites in Kdp, a P-type ATPase of Escherichia coli, were identified by the isolation and characterization of mutants with reduced affinity for K+, its cation substrate. Most of the mutants have an altered KdpA subunit, a hydrophobic subunit not found in other P-type ATPases. Topological analysis of KdpA and the locations of the residues changed in the mutants suggest that KdpA has 10 membrane-spanning segments and forms two separate and distinct sites where K+ is bound. One site is formed by three periplasmic loops of the protein and is inferred to be the site of initial binding. The other site is cytoplasmic. We believe K+ moves from the periplasmic site through the membrane to the cytoplasmic site where it becomes "occluded," i.e. inexchangeable with K+ outside the membrane. Membrane-spanning parts of KdpA probably form the path for transmembrane movement of K+. The kinetics of cation transport in the mutants indicate that each of the two binding sites contributes to the observed Km for cations as well as to the marked discrimination between K+ and Rb+ characteristic of wild-type Kdp. Energy coupling in Kdp, mediated by the KdpB subunit, is performed by a different subunit from the one that mediates transport.
通过分离和鉴定对其阳离子底物K⁺亲和力降低的突变体，确定了大肠杆菌P型ATP酶Kdp中的底物结合位点。大多数突变体的KdpA亚基发生了改变，KdpA是一种在其他P型ATP酶中未发现的疏水亚基。KdpA的拓扑分析以及突变体中发生变化的残基位置表明，KdpA有10个跨膜区段，并形成两个独立且不同的K⁺结合位点。一个位点由该蛋白质的三个周质环形成，推断为初始结合位点。另一个位点在细胞质中。我们认为K⁺从周质位点穿过膜移动到细胞质位点，在那里它被“封闭”，即与膜外的K⁺不可交换。KdpA的跨膜部分可能形成K⁺跨膜移动的路径。突变体中阳离子转运的动力学表明，两个结合位点中的每一个都对观察到的阳离子Km值有贡献，也对野生型Kdp特有的K⁺和Rb⁺之间的显著区分有贡献。由KdpB亚基介导的Kdp中的能量偶联是由与介导转运的亚基不同的亚基进行的。
