Mechanistic analysis of the pump cycle of the KdpFABC P-type ATPase

The high-affinity potassium uptake system KdpFABC is a unique type Ia P-type ATPase, because it separates the sites of ATP hydrolysis and ion transport on two different subunits. KdpFABC was expressed in Escherichia coli. It was then isolated and purified to homogeneity to obtain a detergent-solubilized enzyme complex that allowed the analysis of ion binding properties. The electrogenicity and binding affinities of the ion pump for K+ and H+ were determined in detergent-solubilized complexes by means of the electrochromic styryl dye RH421. Half-saturating K+ concentrations and pK values for H+ binding could be obtained in both the unphosphorylated and phosphorylated conformations of KdpFABC. The interaction of both ions with KdpFABC was studied in detail, and the presence of independent binding sites was ascertained. It is proposed that KdpFABC reconstituted in vesicles translocates protons at a low efficiency opposite from the well-established import of K+ into the bacteria. On the basis of our results, various mechanistic pump cycle models were derived from the general Post–Albers scheme of P-type ATPases and discussed in the framework of the experimental evidence to propose a possible molecular pump cycle for KdpFABC.

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570 Biowissenschaften, Biologie

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DAMNJANOVIC, Bojana, Annemarie WEBER, Meike ODERMATT, Jörg-Christian GREIE, Hans-Jürgen APELL, 2013. Mechanistic analysis of the pump cycle of the KdpFABC P-type ATPase. In: Biochemistry. 2013, 52(33), pp. 5563-5576. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi400729e

BibTex

@article{Damnjanovic2013-08-20Mecha-24433,
  year={2013},
  doi={10.1021/bi400729e},
  title={Mechanistic analysis of the pump cycle of the KdpFABC P-type ATPase},
  number={33},
  volume={52},
  issn={0006-2960},
  journal={Biochemistry},
  pages={5563--5576},
  author={Damnjanovic, Bojana and Weber, Annemarie and Odermatt, Meike and Greie, Jörg-Christian and Apell, Hans-Jürgen}
}

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    <dcterms:abstract xml:lang="eng">The high-affinity potassium uptake system KdpFABC is a unique type Ia P-type ATPase, because it separates the sites of ATP hydrolysis and ion transport on two different subunits. KdpFABC was expressed in Escherichia coli. It was then isolated and purified to homogeneity to obtain a detergent-solubilized enzyme complex that allowed the analysis of ion binding properties. The electrogenicity and binding affinities of the ion pump for K+ and H+ were determined in detergent-solubilized complexes by means of the electrochromic styryl dye RH421. Half-saturating K+ concentrations and pK values for H+ binding could be obtained in both the unphosphorylated and phosphorylated conformations of KdpFABC. The interaction of both ions with KdpFABC was studied in detail, and the presence of independent binding sites was ascertained. It is proposed that KdpFABC reconstituted in vesicles translocates protons at a low efficiency opposite from the well-established import of K+ into the bacteria. On the basis of our results, various mechanistic pump cycle models were derived from the general Post–Albers scheme of P-type ATPases and discussed in the framework of the experimental evidence to propose a possible molecular pump cycle for KdpFABC.</dcterms:abstract>
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