EPR and water proton relaxation rate ( ] / T i ) studies of partially (40%) and "fully" (90%) purified preparations of membrane-bound (Na' + K+) activated ATPase from sheep kidney indicate one tight binding site for Mn" per enzyme dimer, with a dissociation constant (K,, = 0.88 pM) in agreement with the kinetically determined activator constant, identifying this Mn'+-binding site as the active site of the ATPase. Competition studies indicate that Mg" binds at this site with a dissociation constant of 1 mM in agreement with its activator constant. plex with similar high affinities and decrease l / T 1 of water protons due t o a decrease from four to three in the number of rapidly exchanging water protons in the coordination sphere of enzyme-bound Mn". The relative effectiveness of Na' and I K+ in facilitating ternary complex formation with HPOZ-and CH3P03-as a function of pH indicates that Na' induces the phosphate monoanion t o interact with enzyme-bound Mn", while K+ causes the phosphate dianion t o interact with the enzyme-bound Mn2+. Thus protonation of an enzyme-bound phosphoryl group would convert a K+-binding site t o a Na+-binding site. Dissociation constants for K+ and Na', estimated from NMR titrations, agreed with kinetically determined activator constants of these ions consistent with binding t o the active site.
Parallel 32Pi-binding studies show negligible formation (< 7%) of a covalent E-P complex under these conditions, indicating that the NMR method has detected an additional noncovalent intermediate in ion transport. Ouabain, which increases the extent of phosphorylation of the enzyme to 24% at pH 7.5 and t o 106% at pH 6.1, produced further decreases in l / T 1 of water protons. Preliminary 31 Prelaxation studies of CH3P0z-in the presence of ATPase and Mn2+ yield an Mn t o P distance (6.9 * 0.5 8) suggesting a second sphere enzyme-Mn-ligand-CH3POi-complex.
Previous kinetic studies have shown that T l'substitutes for K+ in the activation of the enzyme but competes with Na+ at higher levels. From the paramagnetic effect of Mn2+ at the active site on the enzyme on I/Ti of "'Tl bound at the Na' site, a Mn" t o T1+ distance of 4.0 ? 0.1 8 is calculated, suggesting the sharing of a common ligand atom by Mn" and T l + o n the ATPase. Addition of P. increases this distance to 5.4 8 consistent with the insertion of P between Mn" and Tl'.
Inorganic phosphate and methylphosphonate bind t o the enzyme-Mn' + com-