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

Abstract

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 K0.5 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 K0.5 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. © 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.