The highly conserved extracellular peptide, DSYG(893-896), is a critical structure for sodium pump function.
The peptide sequence DSYG(893-896) of the sheep sodium pump alpha 1 subunit is highly conserved among all K(+)-transporting 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 alpha/beta 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 Ser894-->Asp mutant displayed a threefold higher apparent affinity for K(+) (EC(50) = 1.4 +/- 0.06 mm) than the wild-type enzyme (EC(50) = 3.8 +/- 0.33 mm). In contrast, the mutant Ser894-->Ile had an almost sixfold lower apparent affinity for K(+) (EC(50) = 21.95 +/- 1.41 mm). Mutation of Asp893 or Gly896 produced inactive proteins. When an anti-beta 1 subunit immunoglobulin was used to co-immunoprecipitate the alpha 1 subunit, neither the Gly896-->Arg nor the Gly896-->Ile mutant could be visualized by subsequent probing with an anti-alpha 1 subunit immunoglobulin. On the other hand, co-immunoprecipitation was obtained with the inactive Asp893-->Arg and Asp893-->Glu 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 function of alpha/beta heterodimeric P-type ATPases.
Becker
Susanne
S
Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany.
Schneider
Heike
H
Scheiner-Bobis
Georgios
G
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D010446:Peptide Fragments; D010649:Phenylalanine; D014364:Tryptophan; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine