Evidences for a Sulfhydryl Group in the ATP-Binding Site of (Na+ + K+)-Activated ATPase

5,5′-Dithio-bis(2-nitrobenzoate) inhibited (Na++ K+)-activated ATPase by affecting the Na+-dependent phosphorylation reaction. ATP and ADP but not ITP protected the enzyme at low concentrations against the inactivation. The ATP analogues 6-mercaptopurine riboside-5′-triphosphate and S-(2,4-dinitrophenyl)-6-mercaptopurine riboside-5′-triphosphate inactivated (Na++ K+)-ATPase whereas the respective monophosphates left the enzyme unaffected. S-(2,4-Dinitrophenyl)-6-mercaptopurine triphosphate inactivated the partial reactions of (Na++ K+)-ATPase, i.e. the ADP-binding capacity and the Na+-dependent phosphorylation reaction in parallel with the overall enzymatic activity. ATP protected against the inactivation of (Na++ K+)-ATPase by S-(2,4-dinitrophenyl)-6-mercaptopurine triphosphate. Dithiothreitol hindered the inactivation of (Na++ K+)-ATPase by S-(2,4-dinitrophenyl)-6-mercaptopurine riboside-5′-triphosphate and 6-mercaptopurine riboside-5′-triphosphate. Treatment with dithiothreitol restored the activity of (Na++ K+)-ATPase which had been inactivated by 6-mercaptopurine riboside-5′-triphosphate but it did not restore the activity of that enzyme which was pretreated with S-(2,4-dinitrophenyl)-6-mercaptopurine riboside-5′-triphosphate. S-(2,4-Dinitrophenyl)-6-mercaptopurine riboside-5′-triphosphate inactivated the associated enzymic activites of K+-activated acetylphosphatase and K+-dependent p-nitrophenylphosphatase but more slowly than (Na++ K+)-activated ATPase. It is concluded, that S-(2,4-dinitrophenyl)-6-mercaptopurine riboside-5′-triphosphate and 6-mercaptopurine riboside-5′-triphosphate exert their action on (Na++ K+)-ATPase by reacting with a sulfhydryl group in the ATP-binding site of the enzyme.