title: FITC binding site and p-nitrophenyl phosphatase activity of the Kdp-ATPase of Escherichia coli.
abstract: The KdpFABC complex of Escherichia coli, which belongs to the P-type ATPase family, has a unique structure, since catalytic activity (KdpB) and the capacity to transport potassium ions (KdpA) are located on different subunits. We found that fluorescein 5-isothiocyanate (FITC) inhibits ATPase activity, probably by covalently modifying lysine 395 in KdpB. In addition, we observed that the KdpFABC complex is able to hydrolyze p-nitrophenyl phosphate (pNPP) in a Mg(2+)-dependent reaction. The pNPPase activity is inhibited by FITC and o-vanadate. Low concentrations of ATP (1-30 microM) stimulate the pNPPase activity, while concentrations of >500 microM are inhibitory. This behavior can be explained either by a regulatory ATP binding site, where ATP hydrolysis is required, or by proposing an interactive dimer. The notion that FITC inhibits pNPPase and ATPase activity supports the idea that the catalytic domain of KdpB is much more compact than other P-type ATPases, like Na(+),K(+)-ATPase, H(+),K(+)-ATPase, and Ca(2+)-ATPase.
authors.affiliation: Abteilung Mikrobiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, D-49069 Osnabrück, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Bramkamp
authors.forename: Michael
authors.initials: M
authors.lastname: Gassel
authors.forename: Karlheinz
authors.initials: K
authors.lastname: Altendorf
mesh_terms: D009597:4-Nitrophenylphosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000249:Adenosine Monophosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D014638:Vanadates / Q000737:chemistry
chemical_list: D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D014638:Vanadates; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009597:4-Nitrophenylphosphatase; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine