Photoaffinity Labeling of the Active Site of the Na+/K+-ATPase with 4-Azido-2-nitrophenyl Phosphate

Na+/K+-ATPase will hydrolyze small acylphosphates such as p-nitrophenyl phosphate (pNPP) in addition to ATP and can derive sufficient energy from the hydrolysis of these small molecules to catalyze active ion transport. In this report, 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive analog of pNPP, was used as a probe of the substrate binding site of dog renal Na+/K+-ATPase. ANPP was slowly hydrolyzed by Na+/K+-ATPase with a V max of 0.19 μmol mg-1 min-1 and with an apparent K m of 1.0 mM. The K m for hydrolysis of pNPP was 1.7 mM. ANPP competitively inhibited the hydrolysis of pNPP with a K i of 0.37 mM. Both the ATPase and pNPPase activity of the Na+/K+-ATPase were irreversibly inhibited after photolysis of the enzyme and ANPP with UV light, although neither activity was completely inhibited by up to 200 μM ANPP. Inhibition of activity was prevented by including 0.2 mM ATP in the reaction or by excluding Mg2+ from the photolysis buffer. Photolysis with [32P]ANPP labeled only the α subunit of the Na+/K+-ATPase, and the amount of labeling was substantially reduced by 0.2 mM ATP or in the absence of Mg2+. The stoichiometry of labeling extrapolated to a maximum of about 1.2 nmol/mg of protein at 100% inhibition of Mg2+-dependent activity. Limited proteolytic digestion showed labeling sites on nonoverlapping tryptic peptides derived from the α subunit of Na+/K+-ATPase, and two radiolabeled peptides were purified from an exhaustive tryptic digest of [32P]ANPP-labeled Na+/K+-ATPase. One peptide contained amino acids Met-379 to Lys-406, and the second contained amino acids Ala-655 to Lys-676. Amino acids corresponding to Asn-398 and Pro-668 were missing from the sequences and may represent residues derivatized by ANPP from within the substrate binding site of Na+/K+-ATPase.