title: Genetic evidence for two sequentially occupied K+ binding sites in the Kdp transport ATPase.
abstract: 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.
authors.affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.
authors.forename: E T
authors.initials: ET
authors.lastname: Buurman
authors.forename: K T
authors.initials: KT
authors.lastname: Kim
authors.forename: W
authors.initials: W
authors.lastname: Epstein
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D011188:Potassium / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli; D000252:Calcium-Transporting ATPases; D011188:Potassium