Significance of lysine/glycine cluster structure in gastric H+,K+-ATPase.
Gastric H+,K+-ATPase consists of alpha- and beta-subunits. The catalytic alpha-subunit contains a very unique structure consisting of lysine and glycine clusters, KKK(or KKKK)AG(G/R)GGGK-(K/R)K, in the amino-terminal cytoplasmic region. This structure is well conserved in all gastric H+,K+-ATPases from different animal species, and was postulated to be the site controlling the access of cations (or proton) to its binding site. In this report, we studied the role of this unique structure by expressing several H+,K+-ATPase mutants of the alpha-subunit together with the wild-type beta-subunit in HEK-293 cells. Even after replacing all the positively-charged amino acid residues (six lysines and one arginine) in the cluster with alanine or removing all the glycine residues in the cluster, the mutants preserved the H+,K+-ATPase activity, and showed similar affinity for ATP and K+ as well as similar pH profiles as those of wild-type H+,K+-ATPase, indicating that the cluster is not indispensable for H+,K+-ATPase activity and not directly involved in determination of the affinity for cation (proton).
Asano
S
S
Molecular Genetics Research Center, Toyama Medical and Pharmaceutical University, Toyama, 930-0194 Japan. shinji@ms.toyama-mpu.ac.jp
Miwa
K
K
Yashiro
H
H
Tabuchi
Y
Y
Takeguchi
N
N
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D002412:Cations / Q000493:pharmacokinetics; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D018076:DNA, Complementary; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005753:Gastric Mucosa / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D005998:Glycine / Q000737:chemistry* / Q000235:genetics; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D011188:Potassium / Q000493:pharmacokinetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002412:Cations; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase; D008239:Lysine; D011188:Potassium; D005998:Glycine