Interaction of Na,K-ATPase with modifying ATP analogs and chloromethylphosphonic acid

The interaction of synthetic ATP analogs, containing active groups in the triphosphate moiety and in the 8-position of the nucleotide molecule, with highly purified Na, K-ATPase from the medullar layer of porcine kidney was studied. It was found that 11 out of 17 ATP analogs studied irreversibly inhibit the ATPase activity of the enzyme. The pH optimum of the enzyme inactivation by adenosine-5'-(beta-chloroethylphosphate) and adenosine-5'-(p-fluorosulfonylphenylphosphate) beside the pronounced protective effect of ATP suggests possible covalent blocking of histidine and dicarboxylic amino acid residues in the enzyme active center. The irreversible inhibition of the enzyme by "oxo-ATP" containing aldehyde groups in the modified ribose residue in the presence of sodium borohydride suggests a possible presence of the lysine residue epsilon-amino group in the ATP binding site of the enzyme. Na, K-ATPase was found to possess an inorganic phosphate binding site, which is specifically blocked by chloromethylphosphonic acid. the accessibility of this site for modification depends on ATP, NA+ and K+.