title: Substrate-binding clusters of the K+-transporting Kdp ATPase of Escherichia coli investigated by amber suppression scanning mutagenesis.
abstract: The Kdp-ATPase of Escherichia coli is a four-subunit P-type ATPase that accumulates K(+) with high affinity and specificity. Residues clustered in four regions of the KdpA subunit of Kdp were implicated as critical for K(+) binding from the analysis of mutants with reduced affinity for K(+) (Buurman, E., Kim, K.-T., and Epstein, W. (1995) J. Biol. Chem. 270, 6678-6685). K(+) binding by this pump has been analyzed in detail by site-directed mutagenesis. We have examined 83 of the 557 residues in KdpA, from 11 to 34 residues in each of four binding clusters known to affect K(+) binding. Amber mutations were constructed in a plasmid carrying the kdpFABC structural genes. Transferring these plasmids to 12 suppressor strains, each inserting a different amino acid at amber codons, created 12 different substitutions at the mutated sites. This study delineates the four clusters and confirms that they are important for K(+) affinity but have little effect on the rate of transport. At only 21 of the residues studied did at least three substitutions alter affinity for K(+), an indication that a residue is in or very near a K(+) binding site. At many residues lysine was the only substitution that altered its affinity. The effect of lysine is most likely a repulsive effect of this cationic residue on K(+) and thus reflects the effective distance between a residue and the site of binding or passage of K(+) in KdpA. Once a crystallographic structure of Kdp is available, this measure of effective distance will help identify the path of K(+) as it moves through the KdpA subunit to cross the membrane.
authors: Department of Molecular Genetics and Cell Biology, The University of Chicago, Illinois 60637, USA.
authors: S
authors: S
authors: Dorus
authors: H
authors: H
authors: Mimura
authors: W
authors: W
authors: Epstein
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000838:Anions; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D027682:Cation Transport Proteins; D002412:Cations; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D003062:Codon; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D000838:Anions; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D002412:Cations; D003062:Codon; D029968:Escherichia coli Proteins; D006639:Histidine; D001120:Arginine; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli; C022761:potassium transporting ATPase; D008239:Lysine; D011188:Potassium