title: Different classes of nucleotide binding sites in the (Na+ + K+)-ATPase studied by affinity labeling and nucleotide-dependent SH-group modifications.
abstract: The ATP analog 6-[(3-carboxy-4-nitrophenyl)thiol]-9-beta-D-ribofuranosylpurine 5'-triphosphate (Nbs6ITP) is slowly hydrolyzed at pH 7.4 by the (Na+ + K+)-ATPase, whereas it binds covalently at pH 8.5 and inhibits the enzyme irreversibly. Time courses of irreversible inhibition could only be fitted to a model in which the enzyme can exist in two slowly interchangeable states, one of which is enzymatically active and binds Nbs6ITP first reversibly and then covalently. Arguments that the covalent binding occurs at a low affinity nucleotide binding site are: (a) similarity of the Ki Nbs6ITP for the reversible and the irreversible inhibition and of K0.5 for ATP protection; (b) stoichiometry of covalent Nbs6ITP binding per alpha subunit of 0.8; and (c) change of complex substrate dependence of the enzyme to a Michaelis-Menten type after Nbs6ITP modification. This change in kinetics and the finding that the Nbs6ITP inactivation at a low affinity nucleotide binding site is increased by micromolar ADP concentrations indicates that the (Na+ + K+)-ATPase contains two different nucleotide binding sites. Since studies of nucleotide effects on enzyme inactivation by 5,5'-dithiobis(2-nitrobenzoic acid) did not confirm the hypothesis of an SH-group in a nucleotide binding site, Nbs6ITP may bind to another functional group, e.g. to an OH-group of tyrosine.
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D009579:Nitrobenzoates / Q000494:pharmacology*; D009994:Osmolar Concentration; D011485:Protein Binding; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology*
chemical_list: C016288:6-((3-carboxy-4-nitrophenyl)thio)-9 beta-D-ribofuranosylpurine 5'-triphosphate; D009579:Nitrobenzoates; D013873:Thionucleotides; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D004492:Edetic Acid; D000254:Sodium-Potassium-Exchanging ATPase