Bramkamp Marc, Gassel Michael, Altendorf Karlheinz
Abteilung Mikrobiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, D-49069 Osnabrück, Germany.
Biochemistry. 2004 Apr 20;43(15):4559-67. doi: 10.1021/bi030198a.
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.
大肠杆菌的KdpFABC复合物属于P型ATP酶家族，具有独特的结构，因为催化活性（KdpB）和运输钾离子的能力（KdpA）位于不同的亚基上。我们发现异硫氰酸荧光素（FITC）可能通过共价修饰KdpB中的赖氨酸395来抑制ATP酶活性。此外，我们观察到KdpFABC复合物能够在Mg(2+)依赖的反应中水解对硝基苯磷酸酯（pNPP）。FITC和钒酸盐可抑制pNPP酶活性。低浓度的ATP（1 - 30 microM）刺激pNPP酶活性，而浓度>500 microM则具有抑制作用。这种行为可以通过一个需要ATP水解的调节性ATP结合位点来解释，或者通过提出一个相互作用的二聚体来解释。FITC抑制pNPP酶和ATP酶活性这一观点支持了KdpB的催化结构域比其他P型ATP酶（如Na(+),K(+)-ATP酶、H(+),K(+)-ATP酶和Ca(2+)-ATP酶）更加紧凑的观点。
