PMID:11106663
| Citation |
Dorus, S, Mimura, H and Epstein, W (2001) Substrate-binding clusters of the K+-transporting Kdp ATPase of Escherichia coli investigated by amber suppression scanning mutagenesis. J. Biol. Chem. 276:9590-8 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1074/jbc.M009365200 |
| Keywords |
Adenosine Triphosphatases/metabolism; Anions; Arginine/chemistry; Binding Sites; Carrier Proteins/metabolism; Cation Transport Proteins; Cations; Cell Division; Cell Membrane/metabolism; Codon; Dose-Response Relationship, Drug; Escherichia coli/enzymology; Escherichia coli Proteins; Histidine/chemistry; Kinetics; Lysine/chemistry; Lysine/metabolism; Models, Biological; Mutagenesis, Site-Directed; Mutation; Phenotype; Plasmids/metabolism; Potassium/metabolism; Potassium/pharmacology; Protein Binding; Protein Structure, Tertiary |
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