Inhibition and partial reactions of Na,K-ATPase studied by Fourier transform infrared difference spectroscopy.
Reaction-induced infrared (IR) difference spectroscopy with caged ATP and Na,K-ATPase allows us to differentiate unambiguously between phosphorylated and unphosphorylated states of the enzyme as well as of its ouabain complex. The IR spectral changes upon phosphoenzyme formation are characterized and interpreted. Our results show clearly that high Na(+) concentrations prevent the binding of ouabain with high affinity, which is consistent with the results of a corresponding kinetic study employing spectrofluorimetry and calorimetric titrations. This unexpected antagonism leading to low ouabain affinity is assumed related to a conformation of the protein, induced by low affinity binding of the third Na(+) ion. We thus conclude that not the free enzyme but a phosphorylated state of the reaction cycle preferentially binds ouabain and leads to the loss of hydrolytic activity.
Stolz
Michael
M
Max-Planck Institute of Biophysics, Max von Laue Strasse 3, Frankfurt D-60438, Germany.
Lewitzki
Erwin
E
Mäntele
Werner
W
Barth
Andreas
A
Grell
Ernst
E
D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D010042:Ouabain / Q000378:metabolism; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase