title: Arginine residue at position 573 in Enterococcus hirae vacuolar-type ATPase NtpI subunit plays a crucial role in Na+ translocation.
abstract: The 76-kDa NtpI subunit constitutes the membrane-embedded V(0) moiety of Enterococcus hirae vacuolar type Na+-ATPase with a 16-kDa NtpK hexamer containing Na+ binding sites. In this study, we investigated the role of an arginine residue, which is highly conserved among the corresponding subunits of bacterial vacuolar-type ATPases, at position 573 of NtpI. Substitution of Glu, Leu, or Gln for Arg-573 abolished sodium transport and sodium-stimulated ATP hydrolysis of the enzyme. The conservative replacement of Arg by Lys lowered both activities about one-fifth of those of the wild type enzyme. We have reported previously on ATP-dependent negative cooperativity for Na+ coupling of this enzyme (Murata, T., Kakinuma, Y., and Yamato, I. (2001) J. Biol. Chem. 276, 48337-48340). The negative cooperativity for the Na+ dependence of ATPase activity was weakened by the mutation R573K; the Hill coefficients for the wild type and mutant enzymes at a saturated ATP concentration were 0.22 +/- 0.03 and 0.40 +/- 0.05, respectively. The Hill coefficients of both enzymes at limited ATP concentrations approached 1. These results indicate that NtpI Arg-573 is indispensable for sodium translocation and for the cooperative features of E. hirae vacuolar-type ATPase.
authors: Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
authors: Miyuki
authors: M
authors: Kawano
authors: Kazuei
authors: K
authors: Igarashi
authors: Ichiro
authors: I
authors: Yamato
authors: Yoshimi
authors: Y
authors: Kakinuma
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D001483:Base Sequence; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers; D016983:Enterococcus / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; D017931:DNA Primers; D021122:Protein Subunits; D011510:Proteolipids; D011994:Recombinant Proteins; D001120:Arginine; D012964:Sodium; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases; C021838:sodium-translocating ATPase