IDENTIFICATION OF AN AMINO-ACID IN THE ATP BINDING-SITE OF NA+ K+-ATPASE AFTER PHOTOCHEMICAL LABELING WITH 8-AZIDO-ATP/

[Alpha-P-32]-8-N3-ATP, [2-H-3]-8-N3-ATP, and non-radioactive 8-N3-ATP have been used as photoaffinity probes of the ATP binding site of dog kidney Na+/K+-ATPase. 8-N3-ATP has previously been shown to bind to Na +/K+-ATPase with high affinity, to be a substrate for Na+/K+-ATPase, a nd to inactivate the enzyme upon ultraviolet irradiation [Scheiner-Bob is, G., & Schoner, W. (1985) Eur. J. Biochem. 152, 739-746]. 8-N3-ATP competitively inhibits the high-affinity binding of [2,8-H-3]-ATP to N a+/K+-ATPase with a K(i) of 3.4 muM, which is comparable to the report ed K(D) of 3.1 muM for the binding of 8-N3-ATP to the enzyme. The exte nt of inhibition of ATP hydrolysis by 8-N3-ATP was linearly correlated with the stoichiometry of covalent incorporation of 8-N3-ATP into Na/K+-ATPase up to about 50% inhibition of activity; however, the linkag e between the protein and 8-N3-ATP was unstable, and the maximum incor poration of 8-N3-ATP was less than the nucleotide binding capacity of the protein. After photolysis with ultraviolet light, 8-N3-ATP was spe cifically incorporated into the carboxy-terminal 58-kDa fragment of th e alpha-subunit of Na+/K+-ATPase generated by limited trypsin digestio n in the presence of KCl, and the beta-subunit was not labeled. 8-N3-A TP-labeled Na+/K+-ATPase was digested with trypsin, and a single peak containing the nucleotide was identified after HPLC fractionation of t he digest. The peptide in this peak was purified and sequenced and was found to have the amino acid sequence, u-Ser-Ile-His-Lys-Asn-Pro-Asn- Thr-Ser-Glu-Pro-Arg. This sequence corresponds to amino acids 470-495 of the Na+/K+-ATPase alpha-subunit and is also highly conserved among other P-type ion pumps. The yield from the sequencer at cycle 11, corr esponding to lysine 480, was substantially reduced in the sequence of the photochemically labeled peptide compared to the same sequence of a n unlabeled peptide. These results indicate that 2-N3-ATP labels lysin e 480 of Na+/K+-ATPase from within the ATP binding site of the protein .