PHOTOINACTIVATION OF FLUORESCEIN ISOTHIOCYANATE-MODIFIED NA,K-ATPASE BY 2'(3')-O-(2,4,6-TRINITROPHENYL)8-AZIDOADENOSINE 5'-DIPHOSPHATE - ABOLITION OF E1 AND E2 PARTIAL REACTIONS BY SEQUENTIAL BLOCK OF HIGH ANDLOW-AFFINITY NUCLEOTIDE SITES

The Na,K-ATPase activity of the sodium pump exhibits apparent multisit e kinetics toward ATP, a feature that is inherent to the minimal enzym e unit, the alpha beta protomer. We have argued that this should arise from separate catalytic and noncatalytic sites on the alpha beta prot omer as fluorescein isothiocyanate (FITC) blocks a high affinity ATP s ite on all alpha subunits and yet the modified Na,K-ATPase retains a l ow affinity response to nucleotides (Ward, D. G., and Cavieres, J. D. (1996) J. Biol, Chem. 271, 12317-12321), We now find that 2'(3')-O-(2, 4,6-trinitrophenyl)8-azido-adenosine 5'-diphosphate (TNP-8N(3)-ADP), a high affinity photoactivatable analogue of ATP, can inhibit the K+-ph osphatase activity of the FITC-modified enzyme during assays in dimmed light. The inhibition occurs with a K-i of 140 mu M at 20 mM K+; it r equires the adenine ring as 2'(3')-O-(2,4 B-trinitrophenyl) (TNP)-UDP or TNP-uridine are less potent and 2,4,6-trinitrobenzene-sulfonate is ineffective. Under irradiation with UV light, TNP-8N(3)-ADP inactivate s the K+-phosphatase activity of the fluorescein-enzyme and also its p hosphorylation by [P-32]P-i. The photoinactivation process is stimulat ed by Na+ or Mg2+, and is inhibited by K+ or excess TNP-ADP. In the pr esence of 50 mM Na+ and 1 mM Mg2+, TNP-8N(3)-ADP photoinactivates with a K-0.5 of 15 mu M. Furthermore, TNP-8N(3)-ADP photoinactivates the F ITC-modified, solubilized alpha beta protomers, even more effectively than the membrane-bound fluorescein-enzyme, These results strongly sug gest that catalytic and allosteric ATP sites coexist on the alpha beta protomer of Na,K-ATPase.