Competitive binding of ATP and the fluorescent substrate analogue 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidine) adenosine 5'-triphosphate to the gastric H+,K+-ATPase: evidence for two classes of nucleotide sites.

ATP and the fluorescent substrate analogue TNP-ATP bind competitively to the gastric H,-K-ATPase. Substrate and product completely reverse the fluorescence enhancement caused by TNP-ATP binding to the enzyme. The fluorophore is displaced monophasically from apoenzyme. However, ATP displaces TNP-ATP from the Mg2+-quenched state in two steps of equal amplitude. The midpoints of the titrations differ by more than 2 orders of magnitude. The estimated substrate constants are in reasonable agreement with published Michaelis constants. TNP-ATP is not a substrate for the H,K-ATPase. The fluorophore prevents phosphorylation by ATP and competitively inhibits the K+-stimulated pNPPase and ATPase activities of the enzyme. Ki is approximately the same for both hydrolytic activities and consistent with the Kd of TNP-ATP measured directly. Km for pNPP is 1.48 +/- 0.15 mM. Two Michaelis constants are required to fit the ATPase data: Km1 = 0.10 +/- 0.01 mM and Km2 = 0.26 +/- 0.05 mM.