Binding of the fluorescent substrate analogue 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine 5'-triphosphate to the gastric H+,K(+)-ATPase: evidence for cofactor-induced conformational changes in the enzyme.

TNP-ATP binds to the gastric H,K-ATPase with a 4.6-fold increase in fluorescence intensity and 10-nm blue shift that indicate a relatively hydrophobic protein environment. The fluorescence enhancement saturates and is compatible with binding to a single class of specific nucleotide sites with Kd less than 25 nM and N = 3.4 +/- 0.9 nmol mg-1. Cofactors of the H,K-ATPase affect the fluorescence enhancement. K+ causes a rapid fluorescence quench by binding to a single class of sites with Kd = 3 mM. Mg2+ rapidly and completely reverses the K+ quench and then causes a slow fluorescence quench. The maximum enhancement is approximately halved by either Mg2+ or K+ in titrations with both protein and fluorophore. Therefore, TNP-ATP reports changes in protein environment compatible with cofactor-induced changes in the conformation of the enzyme.