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 (NIPI) 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. Fully 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 alpha-subunit of ATPase. Trypsin treatment of purified alpha-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.