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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
Author(s) -
Puppe Wolfram,
Siebers Annette,
Altendorf Karlheinz
Publication year - 1992
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1992.tb01786.x
Subject(s) - biology , aspartic acid , mutagenesis , site directed mutagenesis , escherichia coli , protein subunit , phosphorylation , biochemistry , atpase , directed mutagenesis , a site , binding site , mutation , amino acid , enzyme , mutant , gene
Summary 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, Gin, 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 Km for K + of the ATPase activity has been increased 30‐fold compared with the wild‐type enzyme.