Thiophosphorylation of [formula omitted] yields an ADP-sensitive phosphointermediate

Abstract

(1) Treatment of (Na + + K +)-ATPase from rabbit kidney outer medulla with the γ- 35 S labeled thio-analogue of ATP in the presence of Na + + Mg 2+ and the absence of K + leads to thiophosphorylation of the enzyme. The K m value for [γ-S]ATP is 2.2 μM and for Na + 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na + concentration, yielding a Hill coefficient n H = 2.6 . (2) The thio-analogue ( K m = 35 μM ) can also support overall (Na + + K +)-ATPase activity, but V max at 37°C is only 1.3 γ mol · (mg protein) − · h −1 or 0.09% of the specific activity for ATP ( K m = 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°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, K m ADP = 48 μM at 0.1 mM ATP ) and is relatively K +-insensitve. The K m 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 E 1-conformation.