Morii Magotoshi, Yamauchi Masashi, Ichikawa Tomohiko, Fujii Takuto, Takahashi Yuji, Asano Shinji, Takeguchi Noriaki, Sakai Hideki
Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
J Biol Chem. 2008 Jun 13;283(24):16876-84. doi: 10.1074/jbc.M800563200. Epub 2008 Apr 9.
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.
在分离的猪胃小泡中，胃H(+)，K(+)-ATP酶显示每摩尔ATP水解可转运2摩尔H(+)。我们研究了H(+)转运机制是否归因于电荷转移和/或水合氢离子（H(3)O(+)）的转移。通过[(18)O]H(2)O的转运，发现每摩尔ATP水解有1.8摩尔水分子被转运。我们对处于E(1)构象的H(+)，K(+)-ATP酶α亚基的三维结构模型进行了分子动力学模拟。它预测存在一条从胞质介质中的水合氢离子到阳离子结合位点2中的Glu-345的电荷转移途径（H(3)O(+)-Lys-164 -Gln-161-Glu-345）。在突变体Q161L、E345L和E345D中未形成电荷转运途径。在突变体K164L中形成了替代途径（H(3)O(+)-Gln-161-Glu-345），在突变体E345Q中形成了（H(3)O(+)-Arg-105-Gln-161-Gln-345）。这些突变体中的H(+)，K(+)-ATP酶活性反映了电荷转移途径的存在与否。我们还发现通过水线和电荷转移途径（H(3)O(+)-Asn-794 -Glu-797）从位点2到位点1存在电荷转移。这些结果表明质子从胞质侧通过电荷转移到位点2和1中的H(2)O，H(2)O来自胞质介质，并且在位点中的H(3)O(+)在从E(1)P到E(2)P的构象转变过程中被转运到管腔中。
