PMID:15078102

Citation	Bramkamp, M, Gassel, M and Altendorf, K (2004) FITC binding site and p-nitrophenyl phosphatase activity of the Kdp-ATPase of Escherichia coli. Biochemistry 43:4559-67
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.
Links	PubMed Online version:10.1021/bi030198a
Keywords	4-Nitrophenylphosphatase/antagonists & inhibitors; 4-Nitrophenylphosphatase/chemistry; 4-Nitrophenylphosphatase/metabolism; Adenosine Diphosphate/chemistry; Adenosine Monophosphate/chemistry; Adenosine Triphosphatases/antagonists & inhibitors; Adenosine Triphosphatases/chemistry; Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/chemistry; Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Cation Transport Proteins/antagonists & inhibitors; Cation Transport Proteins/chemistry; Cation Transport Proteins/metabolism; Conserved Sequence; Enzyme Activation; Escherichia coli Proteins/antagonists & inhibitors; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Fluorescein-5-isothiocyanate/chemistry; Fluorescent Dyes/chemistry; Lysine/chemistry; Molecular Sequence Data; Substrate Specificity; Vanadates/chemistry
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