Involvement of the H3O+-Lys-164 -Gln-161-Glu-345 charge transfer pathway in proton transport of gastric H+,K+-ATPase.
Gastric H(+),K(+)-ATPase is shown to transport 2 mol of H(+)/mol of ATP hydrolysis in isolated hog gastric vesicles. We studied whether the H(+) transport mechanism is due to charge transfer and/or transfer of hydronium ion (H(3)O(+)). From transport of [(18)O]H(2)O, 1.8 mol of water molecule/mol of ATP hydrolysis was found to be transported. We performed a molecular dynamics simulation of the three-dimensional structure model of the H(+),K(+)-ATPase alpha-subunit at E(1) conformation. It predicts the presence of a charge transfer pathway from hydronium ion in cytosolic medium to Glu-345 in cation binding site 2 (H(3)O(+)-Lys-164 -Gln-161-Glu-345). No charge transport pathway was formed in mutant Q161L, E345L, and E345D. Alternative pathways (H(3)O(+)-Gln-161-Glu-345) in mutant K164L and (H(3)O(+)-Arg-105-Gln-161-Gln-345) in mutant E345Q were formed. The H(+),K(+)-ATPase activity in these mutants reflected the presence and absence of charge transfer pathways. We also found charge transfer from sites 2 to 1 via a water wire and a charge transfer pathway (H(3)O(+)-Asn-794 -Glu-797). These results suggest that protons are charge-transferred from the cytosolic side to H(2)O in sites 2 and 1, the H(2)O comes from cytosolic medium, and H(3)O(+) in the sites are transported into lumen during the conformational transition from E(1)PtoE(2)P.
Morii
Magotoshi
M
Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
Yamauchi
Masashi
M
Ichikawa
Tomohiko
T
Fujii
Takuto
T
Takahashi
Yuji
Y
Asano
Shinji
S
Takeguchi
Noriaki
N
Sakai
Hideki
H
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D003600:Cytosol / Q000378:metabolism; D005753:Gastric Mucosa / Q000378:metabolism*; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011522:Protons; D013552:Swine
D011522:Protons; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase