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.
Faller
L D
LD
Department of Medicine, University of California, Los Angeles School of Medicine.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000031:analogs & derivatives; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D005456:Fluorescent Dyes; D005753:Gastric Mucosa / Q000378:metabolism*; D017506:H(+)-K(+)-Exchanging ATPase; D066298:In Vitro Techniques; D007700:Kinetics; D013552:Swine
D001051:Apoenzymes; D005456:Fluorescent Dyes; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D017506:H(+)-K(+)-Exchanging ATPase