Lysine 480 is not an essential residue for ATP binding or hydrolysis by Na,K-ATPase

• Published in: The Journal of biological chemistry Vol. 267; no. 6; pp. 3577 - 3580
• Main Authors: WANG, K, FARLEY, R. A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25.02.1992
• Subjects: Adenosine Triphosphate - metabolism; Analytical, structural and metabolic biochemistry; Animals; ATP; Base Sequence; Biological and medical sciences; Blotting, Western; Dogs; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Hydrolysis; Lysine - genetics; Lysine - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Ouabain - metabolism; Plasmids; Sheep; Sodium-Potassium-Exchanging ATPase - metabolism; tyrosine; Vertebrata; Mammalia; Radiolabelling; Structure activity relation; Enzyme; Site directed mutagenesis; Na,K-ATPase; Binding site; ATP; Aminoacid sequence
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)50562-2

Lysine 480 has been suggested to be essential for ATP binding and hydrolysis by Na,K-ATPase because it is labeled by reagents that are thought to react with the ATPase from within the ATP binding site. In order to test this hypothesis, Lys-480 was changed to Ala, Arg, or Glu by site-directed mutagenesis, and the resultant Na,K-ATPase molecules were expressed in yeast cells. The ATPase activity of each of the mutants was similar to the activity of the wild type enzyme indicating that Lys-480 is not essential for ATP hydrolysis. The binding of [3H]ouabain in both ATP-dependent and inorganic phosphate-dependent reactions was used to determine the apparent affinity of each mutant for ATP or Pi. The K0.5(ATP) for ouabain binding to phosphoenzyme formed from ATP was 1-3 microM for Lys-480, Arg-480, and Ala-480, whereas for Glu-480 the K0.5(ATP) was 18 microM. The K0.5(Pi) for ouabain binding to phosphoenzyme formed from inorganic phosphate was 16-28 microM for Lys-480, Arg-480, and Ala-480, but was 74 microM for Glu-480. The Kd for ouabain binding was similar for both the wild type and mutant Na,K-ATPase molecules (3-6 nM). These data indicate that the substitution of an acidic amino acid for lysine at position 480 appears to reduce the affinity of the Na,K-ATPase for both ATP and phosphate. It is concluded that Lys-480 is not essential for ATP binding or hydrolysis or for phosphate binding by Na,K-ATPase but is likely to be located within the ATP binding site of the Na,K-ATPase.