Presence of a low-affinity nucleotide binding site on the (K + + H +)-ATPase phosphoenzyme

Abstract

The effects of Mg 2+ and nucleotides on the dephosphorylation process of the (K + + H +)-ATPase phosphoenzyme have been studied. Phosphorylation with [ γ- 32P]ATP is stopped either by addition of non-radioactive ATP or by complexing of Mg 2+ with EDTA. The dephosphorylation process is slow and monoexponential when dephosphorylation is initiated with ATP. When phosphorylation is stopped by complexing of Mg 2+ the dephosphorylation process is fast and biexponential. The discrepancy could be explained by a nucleotide mediated inhibition of the dephosphorylation process. The I 0.5 for ATP for this inhibition is 0.1 mM and that for ADP is 0.7 mM, suggesting that a low-affinity binding site is involved. When Mg 2+ is present in millimolar concentrations in addition to the nucleotides the dephosphorylation process is enhanced. Evidence has been obtained that Mg 2+ acts through lowering the affinity for ATP. In contrast to K +, Mg 2+ does not stimulate dephosphorylation in the absence of nucleotides. Mg 2+ and nucleotides show the same interaction in the dephosphorylation process of a phosphoenzyme generated from inorganic phosphate. These findings suggest the presence of a low-affinity nucleotide binding site on the phosphoenzyme, as is found in the (Na + + K +)-ATPase phosphoenzyme. This low-affinity binding site may function as a feed-back mechanism in proton transport.