Mutation of aspartate 804 of Na(+),K(+)-ATPase modifies the cation binding pocket and thereby generates a high Na(+)-ATPase activity.
A series of six different mutants (D804A, D804E, D804G, D804N, D804Q, and D804S) of aspartate 804 present in transmembrane segment 6 of the rat Na(+),K(+)-ATPase alpha(1)-subunit were prepared and expressed in Sf9 cells by use of the baculovirus expression system. In contrast to the wild-type enzyme all mutants except D804Q showed a very high Na(+)-ATPase activity, which was hardly further stimulated by the addition of K(+). The ATPase activity of the mutants was already nearly maximal at 10 microM ATP and most of them could be phosphorylated in the absence of Na(+) at pH 6.0 and 21 degrees C, suggesting that they strongly prefer the E(1) over the E(2) conformation. However, Na(+) dose-dependently lowered the steady-state phosphorylation level, as a consequence of the increased affinity for Na(+) in the dephosphorylation reaction of the mutants compared to the wild-type enzyme. Conversely, the affinity for K(+) in the dephosphorylation reaction was decreased for the mutants as compared to that for the wild-type enzyme. When the pH was increased or the temperature was decreased, the phosphorylation level of the mutants decreased and the Na(+) activation in the phosphorylation reaction became apparent. It is concluded that upon mutation of aspartate 804 the affinity of the cation-binding pocket is changed relatively in favor of Na(+) instead of K(+), as a consequence of which the enzyme has obtained a preference for the E(1) conformation.
Department of Biochemistry, Institute of Cellular Signaling, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
J B
JB
Koenderink
H G
HG
Swarts
H P
HP
Hermsen
P H
PH
Willems
J J
JJ
De Pont
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D016367:Baculoviridae; D001665:Binding Sites; D027682:Cation Transport Proteins; D002414:Cations, Monovalent / Q000378:metabolism; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology / Q000821:virology
D027682:Cation Transport Proteins; D002414:Cations, Monovalent; D011994:Recombinant Proteins; D001224:Aspartic Acid; D010042:Ouabain; D012964:Sodium; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium