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,, Rim, K,-T,, and Epstein, W, (1995) J, Biol Chem, 270, 6678-66851) 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 Kdpk 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.
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 2001|