Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate.
1) Treatment of (Na+ + K+)-ATPase from rabbit kidney outer medulla with the gamma-35S labeled thio-analogue of ATP in the presence of Na+ + Mg2+ and the absence of K+ leads to thiophosphorylation of the enzyme. The Km value for [gamma-S]ATP is 2.2 microM and for Na+ 4.2 mM at 22 degrees C. Thiophosphorylation is a sigmoidal function of the Na+ concentration, yielding a Hill coefficient nH = 2.6. (2) The thio-analogue (Km = 35 microM) can also support overall (Na+ + K+)-ATPase activity, but Vmax at 37 degrees C is only 1.13 mumol X (mg protein)-1 X h-1 or 0.09% of the specific activity for ATP (Km = 0.43 mM). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0 degree C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5-6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s-1 vs. 180 s-1), spontaneous dethiophosphorylation (0.04 s-1 vs. 0.5 s-1) and K+-stimulated dethiophosphorylation (0.54 s-1 vs. 230 s-1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s-1, Km ADP = 48 microM at 0.1 mM ATP) and is relatively K+-insensitive. The Km for K+ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75-90% in the ADP-sensitive E1-conformation.
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D006868:Hydrolysis; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D010766:Phosphorylation; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism
D013438:Sulfhydryl Compounds; D013873:Thionucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase