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The peptide sequence DSYG(893–896) of the sheep sodium pumpa1 subunit is highly conserved among all K + -trans-porting P-type ATPases. To obtain information about its function, single mutations were introduced and the mutants were expressed in yeast and analysed for enzymatic activity, ion recognition, and a/bsubunit interactions. Mutants of Ser894 or Tyr895 were all active. Conservative phenylalan-ine and tryptophan mutants of Tyr895 displayed properties that were similar to the properties of the wild-type enzyme | Eur. J. Biochem. 271 3821-3831 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.04305.x The highly conserved extracellular peptide DSYG 893-896 is a critical structure for sodium pump function Susanne Becker Heike Schneider and Georgios Scheiner-Bobis Institut fur Biochemie und Endokrinologie Fachbereich Veterinarmedizin Justus-Liebig-Universitat Giessen Germany The peptide sequence DSYG 893-896 of the sheep sodium pump a1 subunit is highly conserved among all K -trans-porting P-type ATPases. To obtain information about its function single mutations were introduced and the mutants were expressed in yeast and analysed for enzymatic activity ion recognition and a b subunit interactions. Mutants of Ser894 or Tyr895 were all active. Conservative phenylalanine and tryptophan mutants of Tyr895 displayed properties that were similar to the properties of the wild-type enzyme. Replacement of the same amino acid by cysteine however produced heat-sensitive enzymes indicating that the aromatic group contributes to the stability of the enzyme. Mutants of the neighbouring Ser894 recognized K with altered apparent affinities. Thus the Ser894fiAsp mutant displayed a threefold higher apparent affinity for K EC50 1.4 0.06 mM than the wild-type enzyme EC50 3.8 0.33 mM . In contrast the mutant Ser894fiIle had an almost sixfold lower apparent affinity for K EC50 21.95 1.41 mM . Mutation of Asp893 or Gly896 produced inactive proteins. When an anti-b1 subunit immunoglobulin was used to co-immunoprecipitate the a1 subunit neither the Gly896fiArg nor the Gly896fiIle mutant could be visualized by subsequent probing with an anti-a1 subunit immunoglobulin. On the other hand co-immunoprecipitation was obtained with the inactive Asp893fiArg and Asp893fiGlu mutants. Thus it might be that Asp893 is involved in enzyme conformational transitions required for ATP hydrolysis and or ion translocation. The results obtained here demonstrate the importance of the highly conserved peptide DSYG 893-896 for the
