Magnetic resonance and kinetic studies of the mechanism of membrane‐bound sodium and potassium ION‐activated adenosine triphosphatase

Abstract

EPR and water proton relaxation rate (1/T₁) 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 Mn²⁺ per enzyme dimer, with a dissociation constant (K_D = 0.88 μM) in agreement with the kinetically determined activator constant, identifying this Mn²⁺-binding site as the active site of the ATPase. Competition studies indicate that Mg²⁺ binds at this site with a dissociation constant of 1 mM in agreement with its activator constant. Inorganic phosphate and methylphosphonate bind to the enzyme-Mn²⁺ complex with similar high affinities and decrease l/T₁ 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 Mn²⁺. The relative effectiveness of Na⁺ and K⁺ in facilitating ternary complex formation with HPO and CH₃PO as a function of pH indicates that Na⁺ induces the phosphate monoanion t o interact with enzyme-bound Mn²⁺, while K⁺ causes the phosphate dianion to interact with the enzyme-bound Mn²⁺. Thus protonation of an enzyme-bound phosphoryl group would convert a K⁺-binding site to 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 ³²P_i-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 ³¹P-relaxation studies of CH₃PO in the presence of ATPase and Mn²⁺ yield an Mn to P distance (6.9 ± 0.5 Å) suggesting a second sphere enzyme-Mn-ligand-CH₃PO complex. Previous kinetic studies have shown that T1⁺ 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/T₁ of ²⁰⁵T1 bound at the Na⁺ site, a Mn²⁺ to T1⁺ distance of 4.0 ± 0.1 Å is calculated, suggesting the sharing of a common ligand atom by Mn²⁺ and T1⁺ on the ATPase. Addition of P. increases this distance to 5.4 Å consistent with the insertion of P between Mn²⁺ and T1⁺. These results are consistent with a mechanism for the \documentclass{article}\pagestyle{empty}\begin{document}$ (\mathop {\rm N}\limits^{\rm i} {\rm a}^{\rm + } {\rm + K}^ +) $\end{document}-ATPase and for ion transport in which the ionization state of Pi at a single enzyme active site controls the binding and transport of Na⁺ and K⁺, and indicate that the transport site for monovalent cations is very near the catalytic site of the ATTase. Our mechanism also accounts for the order of magnitude weaker binding of Na⁺ compared to K⁺.