Structure and function of the Kdp-ATPase of Escherichia coli.
The kdpFABC operon of Escherichia coli consists of the four structural genes kdpF, kdpA, kdpB, and kdpC. Expression of the kdpF gene was demonstrated using minicells of E. coli. In addition, it was shown that the KdpF subunit remains associated with the purified complex. Although KdpF is not essential in vivo, the purified complex lacking KdpF exhibits hardly any K(+)-stimulated ATPase activity. This clearly demonstrates that the KdpF subunit is stabilizing the transport complex. Charge translocation by the purified Kdp-ATPase was measured with the potential-sensitive dye DiSC3(5) using proteoliposomes. Upon addition of ATP a fluorescence quench was observed indicating the buildup of a negative potential inside the proteoliposomes. Using the Kdp-ATPase derived from a mutant strain, in which the K(m) value for K+ (1,2 mM) was almost identical to that of Rb+ (1.4 mM), the same fluorescence quench was observed when K+ or Rb+ were present in the lumen of the proteoliposomes. These data clearly indicate that the Kdp-ATPase transports K+ in an electrogenic manner. In order to identify the binding site(s) for the inhibitor concanamycin A within the Kdp complex, concanamycin A was synthesized. Using this compound labeling of KdpA and KdpB, but not of KdpC, could be shown with the purified complex. When everted vesicles were used only KdpB could be labeled.
Altendorf
K
K
Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, Germany. altendorf@sfbbio1.biologie.uni-osnabrueck.de
Gassel
M
M
Puppe
W
W
Möllenkamp
T
T
Zeeck
A
A
Boddien
C
C
Fendler
K
K
Bamberg
E
E
Dröse
S
S
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D027682:Cation Transport Proteins; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D018942:Macrolides; D019920:Photoaffinity Labels; D011188:Potassium / Q000378:metabolism; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D018942:Macrolides; D019920:Photoaffinity Labels; C036978:concanamycin A; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli; D011188:Potassium