Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase. Their relevance to phosphatase activity.
The K+-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na+ + K+)-ATPase from pig kidney shows substrate inhibition (Ki about 9.5 mM at 2.1 mM Mg2+). 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 Mg2+, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb+ from the Rb+ occluded unphosphorylated enzyme. With no Mg2+ 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 MgCl2, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E1 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 E1 state is the basis for substrate inhibition of the p-nitrophenylphosphatase activity of(Na+ + K+)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.
D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine; D014357:Trypsin / Q000494:pharmacology
D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D011189:Potassium Chloride; D012964:Sodium; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium