E2P phosphoforms of Na,K-ATPase. II. Interaction of substrate and cation-binding sites in Pi phosphorylation of Na,K-ATPase.

In this investigation the effects of alkali cations on the transient kinetics of Na,K-ATPase phosphoenzyme formation from either ATP (E2P) or Pi (E'2P) were characterized by chemical quench methods as well as by stopped-flow RH421 fluorescence experiments. By combining the two methods it was possible to characterize the kinetics of Na, K-ATPase from two sources, shark rectal glands and pig kidney. The rate of the spontaneous dephosphorylation of E2P and E'2P was identical with a rate constant of about 1.1 s-1 at 20 degreesC. However, whereas dephosphorylation of E2P formed from ATP was strongly stimulated by K+, dephosphorylation of E'2P formed from Pi in the absence of alkali cations was K+-insensitive, although in pig renal enzyme K+ binding to E'2P could be demonstrated with RH421 fluorescence. It appears, therefore, that in pig kidney enzyme the rapid binding of K+ to E'2P was followed by a slow transition to a nonfluorescent form. For shark enzyme the K+-induced decrease of RH421 fluorescence of Pi phosphorylated enzyme was due to K+ binding to the dephosphoenzyme (E1), thus shifting the equilibrium away from E'2P. When Pi phosphorylation was performed with enzyme equilibrated with K+ or its congeners Tl+, Rb+, and Cs+ but not with Na+ or Li+, both the phosphorylation and the dephosphorylation rates were considerably increased. This indicates that binding of cations modifies the substrate site in a cation-specific way, suggesting an allosteric interaction between the conformation of the cation-binding sites and the phosphorylation site of the enzyme.