FITC binding site and p-nitrophenyl phosphatase activity of the Kdp-ATPase of Escherichia coli

Autor(en): Bramkamp, M
Gassel, M
Altendorf, K 
Stichwörter: BACILLUS-SUBTILIS; Biochemistry & Molecular Biology; CONFORMATIONAL-CHANGES; FLUORESCEIN ISOTHIOCYANATE; HIGH-AFFINITY; NA+/K+-ATPASE; NUCLEOTIDE SITES; PHOSPHORYLATED INTERMEDIATE; SARCOPLASMIC-RETICULUM CA2+-ATPASE; SUBSTRATE HYDROLYSIS; WILSONS-DISEASE PROTEIN
Erscheinungsdatum: 2004
Herausgeber: AMER CHEMICAL SOC
Journal: BIOCHEMISTRY
Volumen: 43
Ausgabe: 15
Startseite: 4559
Seitenende: 4567
Zusammenfassung: 
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 Mg2+-dependent reaction. The pNPPase activity is inhibited by FITC and o-vanadate. Low concentrations of ATP (1-30 muM) stimulate the pNPPase activity, while concentrations of >500 muM 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 Ca2+-ATPase.
ISSN: 00062960
DOI: 10.1021/bi030198a

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