Role of the charged amino acid residues in the cytoplasmic loop between putative transmembrane segments 6 and 7 of Na+-ATPase of an alkaliphilic bacterium, Exiguobacterium aurantiacum.
ATPase activity of the membrane-bound Na(+)-ATPase of an alkaliphilic bacterium, Exiguobacterium aurantiacum, was measured in various concentrations of NaCl. Hill plot analysis showed a Hill number of 1.7 with 5.2 mM as the K(0.5) value for Na(+). When the site-directed mutagenesis of seven charged amino acid residues in the cytoplasmic loop (L6/7) between putative transmembrane segments 6 and 7 of the enzyme was conducted, all the mutated enzymes exhibited Hill numbers close to that of the wild-type enzyme (WT). When reconstituted with lecithin, all the mutants exhibited Na(+)-transport activity. While alanine substitution for several residues gave some significant effects on the enzyme function, the most remarkable effect was observed in the substitution for Glu-733. The K(0.5) value of E733A for Na(+) was 83.2 mM. The mutant exhibited only 8.5% of the ATPase activity and 54.0% of the energy-coupling efficiency for Na(+) transport as compared with those of WT, respectively. Drastic decreases of apparent affinity for Na(+) and energy efficiency of ion transport were also observed in E733K and E733T, respectively.
Takemura
Youhei
Y
Department of Chemistry, Faculty of Science, Chiba University, Chiba, Japan.
Tamura
Naoki
N
Imamura
Maya
M
Koyama
Noriyuki
N
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000378:metabolism
D000596:Amino Acids; D027682:Cation Transport Proteins; D000255:Adenosine Triphosphate; D012964:Sodium; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase