THE PHOSPHORYLATION SITE OF THE KDP-ATPASE OF ESCHERICHIA-COLI - SITE-DIRECTED MUTAGENESIS OF THE ASPARTIC-ACID RESIDUES 300 AND 307 OF THE KDPB SUBUNIT

Abstract

The potassium-translocating Kdp-ATPase of Escherichia coli shares common functional properties with eukaryotic P-type ATPases. The KdpB subunit has been identified as the catalytic subunit forming the phosphorylated intermediate. Substitution of Asp-307 in KdpB by Glu, Asn, Gln, Tyr, His, Ala or Ser by site-directed mutagenesis and the subsequent transfer of the point mutations to the chromosome revealed that the mutants were not functioning with respect to cell growth at low K+ concentrations and ATPase activity as well as phosphorylation capacity of the purified Kdp complex. These findings indicate that Asp-307 in KdpB is the phosphorylation site of the Kdp-ATPase. In contrast, replacement of the close but non-conserved Asp-300 by Asn or Glu has no immediate influence on the enzyme functions tested. However, the K(m) for K+ of the ATPase activity has been increased 30-fold compared with the wild-type enzyme.