GENETIC-EVIDENCE FOR 2 SEQUENTIALLY OCCUPIED K+ BINDING-SITES IN THE KDP TRANSPORT ATPASE

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 K-m 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.