Potassium- p-nitrophenyl phosphate interactions with (Na + + K +)-ATPase their relevance to phosphatase activity

Abstract

The K +-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na + + K +)-ATPase from pig kidney shows substrate inhibition ( K i about 9.5 mM at 2.1 mM Mg 2+). Potassium antagonizes and sodium favours this inhibition. In addition, K + reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K + activation. In the absence of Mg 2+, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb + from the Rb + occluded unphosphorylated enzyme. With no Mg 2+ and with 0.5 mM KCl, trypsin inactivation of (Na + + K +)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl 2, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E 1 enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E 1 state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na + + K +)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.