Molecular Microbiology (1992) 6(23), 3511-3520
The phosphorylation site of the Kdp-ATPase of Escherichia co//: site-directed mutagenesis of the aspartic acid residues 300 and 307 of the KdpB subunit Wolfram Puppe, Annette Siebers and Karlheinz Altendorf* Universitat Osnabruck, Fachbereich Biologie/Chemie, Arbeitsgruppe Mikrobiologie, Barbarastrasse 11, D-4500 Osnabruck, Germany. 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 Asp307 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 K^, for K* of the ATPase activity has been increased 30-fold compared with the wildtype enzyme.
Introduction The K*-translocating Kdp-ATPase of Escherichia coii is a high-affinity, inducible K^-uptake system (with a K^ for transport of about 2 jiM), the expression of which is turgor-regulated (Epstein. 1986; Epstein etai, 1990). It is expressed as an emergency system under K*-limiting growth conditions when the high-capacity Trk system {/
The phosphorylation site of the Kdp-ATPase of Escherichia co//: site-directed mutagenesis of the aspartic acid residues 300 and 307 of the KdpB subunit Wolfram Puppe, Annette Siebers and Karlheinz Altendorf* Universitat Osnabruck, Fachbereich Biologie/Chemie, Arbeitsgruppe Mikrobiologie, Barbarastrasse 11, D-4500 Osnabruck, Germany. 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 Asp307 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 K^, for K* of the ATPase activity has been increased 30-fold compared with the wildtype enzyme.
Introduction The K*-translocating Kdp-ATPase of Escherichia coii is a high-affinity, inducible K^-uptake system (with a K^ for transport of about 2 jiM), the expression of which is turgor-regulated (Epstein. 1986; Epstein etai, 1990). It is expressed as an emergency system under K*-limiting growth conditions when the high-capacity Trk system {/
