PHOSPHATE BINDING AND ATP-BINDING SITES COEXIST IN NA+/K+-TRANSPORTING ATPASE, AS DEMONSTRATED BY THE INACTIVATING MGPO4 COMPLEX ANALOG CO(NH3)4PO4

Tetrammine cobalt(III) phosphate [Co(NH3)4PO4] inactivates Na+/K+-ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1): [GRAPHICS] The inactivation rate constant k2 for the formation of a stable E2' . Co(NH3)4PO4 at 37-degrees-C was 0.057 min-1, the dissociation constant, K(d) = 300-mu-M. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5'-[beta,gamma-methylene]triphosphate competed with Co(NH3)4PO4 for its binding site with K(s) = 0.41 mM and 5 mM, respectively. MgPO4 competed with Co(NH3)4PO4 linearly, with K(s) = 50-mu-M, as did phosphate (K(s) = 16 mM) and Mg2+ (K(s) = 160-mu-M). It is concluded that the MgPO4 analogue binds to the MgPO4-binding subsite of the low-affinity ATP-binding site (of the E2 conformation). Also, Na+ (K(s) = 860-mu-M) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na+ against the inactivation by Co(NH3)4PO4, apparent affinities of K+ for the free enzyme of 41-mu-M, and for the E . Co(NH3)4PO4 complex of 720-mu-M, were calculated. Binding of Co(NH3)4PO4 to the enzyme inactivated Na+/K+-ATPase and K+-activated phosphatase, and, moreover, prevented the occlusion of Rb-86+; however, the activity of the Na+-ATPase, the phosphorylation capacity of the high-affinity ATP-binding site and the ATP/ADP-exchange reaction remained unchanged. With Co(NH3)4 (PO4)-P-32 a binding capacity of 135 pmol/unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with Co(NH3)4 (PO4)-P-32 or the P-32-labelled tetramminecobalt ATP {[gamma-P-32]Co(NH3)4ATP} at the low-affinity ATP-binding site, allowed (independent of the purity of the Na+/K+-ATPase preparation) a further incorporation of radioactivity from P-32-labelled tetraaquachromium(III) ATP ([gamma-P-32]CrATP) to the high-affinity ATP-binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K+-ATPase by [gamma-P-32]CrATP prevented the binding of Co(NH3)4 (PO4)-P-32 or [gamma-P-32]Co(NH3)4ATP to the enzyme. [gamma-P-32]CO(NH3)4ATP and Co(NH3)4 (PO4)-P-32 are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (alpha-beta)2-diprotomer, which change their interactions during the Na+/K+-pumping process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action.