Modification of lysine 501 in Na,K-ATPase reveals coupling between cation occupancy and changes in the ATP binding domain

Treatment of the canine renal Na,K-ATPase with N-(2-nitro-4-isothiocyanophenyl)-imidazole (NIFI) results in irreversible loss of enzymatic activity. The reactivity of the protein toward the probe is about 10-fold greater in the E₁.·Na or sodium-bound form than when it is in the E₂·K or potassium-bound form, tully inactivated enzyme does not bind ATP but binds sodium and potassium and undergoes the major enzyme conformational transitions (Ellis-Davies G. C. R., and Kaplan, J. H. (1990) J. Biol. Chem. 265, 20570-20576). Labeling of the sodium pump protein with [³H] NIPI in either the E₁·Na or E₂·K conformations results in the covalent incorporation of one molecule of probe per α-subunit of ATPase. Trypsin treatment of purified α-subunit, and separation of the digest using reverse-phase high performance liquid chromatography, yields five major radioactive fragments in each case. Amino acid sequencing indicates that only one residue, lysine 501, is labeled by NIPI. This suggests that the integrity of the domain containing Lys⁵⁰¹ is essential for both high and low affinity binding of ATP by the Na,K-ATPase. Furthermore, the spatial organization of the protein close to lysine 501 is changed in the E¹·Na and E²·K form of the enzyme. This change is reflected in the greater reactivity of lysine 501 in the E₁·Na conformation and indicates that the binding of either sodium or potassium ions to their sites on the sodium pump has differential effects on the nucleotide binding domain containing lysine 501.