Characterization of the phosphorylated intermediate of the K+-translocating Kdp-ATPase from Escherichia coli.
During ATP hydrolysis the K+-translocating Kdp-ATPase from Escherichia coli forms a phosphorylated intermediate as part of the catalytic cycle. The influence of effectors (K+, Na+, Mg2+, ATP, ADP) and inhibitors (vanadate, N-ethylmaleimide, bafilomycin A1) on the phosphointermediate level and on the ATPase activity was analyzed in purified wild-type enzyme (apparent Km = 10 microM) and a KdpA mutant ATPase exhibiting a lower affinity for K+ (Km = 6 mM). Based on these data we propose a minimum reaction scheme consisting of (i) a Mg2+-dependent protein kinase, (ii) a Mg2+-dependent and K+-stimulated phosphoprotein phosphatase, and (iii) a K+-independent basal phosphoprotein phosphatase. The findings of a K+-uncoupled basal activity, inhibition by high K+ concentrations, lower ATP saturation values for the phosphorylation than for the overall ATPase reaction, and presumed reversibility of the phosphoprotein formation by excess ADP indicated similarities in fundamental principles of the reaction cycle between the Kdp-ATPase and eukaryotic E1E2-ATPases. The phosphoprotein was tentatively characterized as an acylphosphate on the basis of its alkali-lability and its sensitivity to hydroxylamine. The KdpB polypeptide was identified as the phosphorylated subunit after electrophoretic separation at pH 2.4, 4 degrees C of cytoplasmic membranes or of purified ATPase labeled with [gamma-32P]ATP.
Siebers
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A
Arbeitsgruppe Mikrobiologie, Universität Osnabrück, Federal Republic of Germany.
Altendorf
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K
D000227:Adenine Nucleotides / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D027682:Cation Transport Proteins; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology
D000227:Adenine Nucleotides; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D004492:Edetic Acid; D012964:Sodium; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli; D011188:Potassium