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

In this investigation the effects of alkali cations on the transient kineti cs of Na,K-ATPase phosphoenzyme formation from either ATP (E2P) or P-i (E'P -2) were characterized by chemical quench methods as well as by stopped-flo w RH421 fluorescence experiments. By combining the two methods it was possi ble to characterize the kinetics of Na,K-ATPase from two sources, shark rec tal glands and pig kidney. The rate of the spontaneous dephosphorylation of E2P and E'P-2 was identical with a rate constant of about 1.1 s(-1) st 20 degrees C. However, whereas dephospharylation of E2P formed front ATP was s trongly stimulated by K+, dephosphorylation of E'P-2 formed from P-i in the absence of alkali cations was K+-insensitive, although in pig renal enzyme K+ binding to E'P-2 could be demonstrated with RH421 fluorescence. It appe ars, therefore, that in pig kidney enzyme the rapid binding of K+ to E'P-2 was followed by a slow transition to a nonfluorescent form. For shark enzym e the K+-induced decrease of RH421 fluorescence of P-i phosphorylated enzym e was due to K+ binding to the dephosphoenzyme (E-l), thus shifting the equ ilibrium away from E'P-2. When P-i phosphorylation was performed with enzym e equilibrated with K+ or its congeners Tl+, Rb+, and Cs+ but not with Naor Li+, both the phosphorylation and the dephosphorylation rates were consi derably increased. This indicates that binding of cations modifies the subs trate site in a cation-specific way, suggesting an allosteric interaction b etween the conformation of the cation-binding sites and the phosphorylation site of the enzyme.