Mutational analysis of the putative K(+)-binding site on the fourth transmembrane segment of the gastric H(+),K(+)-ATPase.
By means of a functional expression system and site-directed mutagenesis, we analyzed the role of the putative K(+)-binding site, Glu-345, located in the fourth transmembrane segment of the gastric H(+),K(+)-ATPase alpha-subunit. In the present study, we used several mutants, with alanine, isoleucine, leucine, glutamine, valine, lysine, and aspartic acid instead of Glu-345, and analyzed the H(+),K(+)-ATPase partial reactions of the mutants to determine the precise role of this residue. All the mutants except E345Q exhibited no H(+),K(+)-ATPase activity. The E345Q mutant showed 3-times higher affinity for ATP. This mutation shifted the optimum pH toward a more alkaline one. The E345A, E345I, E345L, E345V as well as E345Q mutants were phosphorylated with ATP as in the case of the wild-type H(+),K(+)-ATPase, whereas the E345K mutant was not phosphorylated. The E345Q mutant was dephosphorylated in the presence of K(+), but its affinity for K(+) was significantly lower than that of the wild type. The E345A, E345I, E345L, and E345V mutants did not exhibit sensitivity to K(+) in the dephosphorylation step below 3 mM K(+). Therefore, Glu-345 is important for the conformational change induced by K(+), especially in the dephosphorylation step in which K(+) reacts with the enzyme from the luminal side with high affinity and accelerates the release of inorganic phosphate. The glutamic acid in the fourth transmembrane segment is conserved, and was found to be involved in the cation-induced conformational change in H(+),K(+)-ATPase as well as Na(+),K(+)-ATPase and Ca(2+)-ATPase, however, the precise roles of the side chain in the function were different.
Asano
S
S
Molecular Genetics Research Center and Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-0194, Japan. shinji@ms.toyama-mpu.ac.jp
Furumoto
R
R
Tega
Y
Y
Matsuda
S
S
Takeguchi
N
N
D000255:Adenosine Triphosphate / Q000737:chemistry; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004252:DNA Mutational Analysis; D005753:Gastric Mucosa / Q000201:enzymology*; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D014162:Transfection
D000596:Amino Acids; D008565:Membrane Proteins; D005973:Glutamine; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium