Imagawa Toshiaki, Kaya Shunji, Taniguchi Kazuya
Biological Chemistry, Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.
J Biol Chem. 2003 Dec 12;278(50):50283-92. doi: 10.1074/jbc.M309833200. Epub 2003 Sep 30.
A highly conserved amino acid sequence 442GDASE446 in the ATP binding pocket of rat Na/K-ATPase was mutated, and the resulting proteins, G442A, G442P, D443A, S445A, and E446A, were expressed in HeLa cells to investigate the effect of individual ligands on Na/K-ATPase. The apparent Km for the high and low affinity ATP effects was estimated by ATP concentration dependence for the formation of the Na-dependent phosphoenzyme (Kmh) and Na/K-ATPase activity (Kml). The apparent Km for p-nitrophenylphosphate (pNPP) for K-dependent-pNPPase (KmP) and its inhibition by ATP (Ki,0.5) and the apparent Km for Mg2+, Na+, K+, and vanadate in Na/K-ATPase were also estimated. For all the mutants, the value for ATP was approximately 2-10-fold larger than that of the wild type. While the turnover number for Na/K-ATPase activity were unaffected or reduced by 20 approximately 50% in mutants G442(A/P) and D443A. Although both affinities for ATP effects were reduced as a result of the mutations, the ratio, Kml Kmh, for each mutant was 1.3 approximately 3.7, indicating that these mutations had a greater impact on the low affinity ATP effect than on the high affinity effect. Each KmP value with the turnover number suggests that these mutations favor the binding of pNPP over that of ATP. These data and others indicate that the sequence 442GDASE446 in the ATP binding pocket is an important motif that it is involved in both the high and low affinity ATP effects rather than in free Mg2+, Na+, and K+ effects.
对大鼠钠钾ATP酶ATP结合口袋中高度保守的氨基酸序列442GDASE446进行突变，然后将产生的蛋白质G442A、G442P、D443A、S445A和E446A在HeLa细胞中表达，以研究单个配体对钠钾ATP酶的影响。通过钠依赖性磷酸酶（Kmh）形成和钠钾ATP酶活性（Kml）的ATP浓度依赖性来估计高亲和力和低亲和力ATP效应的表观Km。还估计了对硝基苯磷酸酯（pNPP）对钾依赖性pNPP酶的表观Km（KmP）及其被ATP抑制的情况（Ki,0.5），以及钠钾ATP酶中镁离子、钠离子、钾离子和钒酸盐的表观Km。对于所有突变体，ATP的值比野生型大约大2至10倍。虽然钠钾ATP酶活性的周转数在突变体G442(A/P)和D443A中未受影响或降低了约20%至50%。尽管由于突变，两种对ATP效应的亲和力都降低了，但每个突变体的Kml/Kmh比值为1.3至3.7，表明这些突变对低亲和力ATP效应的影响比对高亲和力效应的影响更大。每个KmP值与周转数表明，这些突变有利于pNPP而非ATP的结合。这些数据及其他数据表明，ATP结合口袋中的序列442GDASE446是一个重要基序，它参与高亲和力和低亲和力ATP效应，而非游离镁离子、钠离子和钾离子效应。
