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.
Department of Biology, University of Konstanz, 78464 Konstanz, Germany.
Bojana
B
Damnjanovic
Annemarie
A
Weber
Meike
M
Potschies
Jörg-Christian
JC
Greie
Hans-Jürgen
HJ
Apell
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D027682:Cation Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D017136:Ion Transport; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D011726:Pyridinium Compounds / Q000737:chemistry; D012964:Sodium / Q000378:metabolism; D013343:Styrenes / Q000737:chemistry; D013816:Thermodynamics
D027682:Cation Transport Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D008081:Liposomes; D021122:Protein Subunits; D011522:Protons; D011726:Pyridinium Compounds; D013343:Styrenes; C070534:RH 421; D000255:Adenosine Triphosphate; D012964:Sodium; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli; D011188:Potassium