How do MgATP analogues differentially modify high‐affinity and low‐affinity ATP binding sites of Na+/K+ ‐ATPase?

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Abstract
The exchange-inert tetra-amino-chromium complex of ATP [Cr(NH3)4-ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na+/K+-ATPase slowly by interacting with the high-affinity ATP binding site. The inactivation proceeded at 37.degree. C with an inactivation rate constant of 1.34 .times. 10-3 min-1 and with a dissociation constant of 0.62 .mu.M. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K+-ATPase by Cr(NH3)4-ATP, and its H2O-substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4-ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4-ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme''s active-site residues does not seem to play a significant role in the inactivation of Na+/K+-ATPase by Cr(III)-ATP complexes. Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 .mu.M. The observed inactivation rate constant of 2.11 .times. 10-3 min-1 became higher when Na+ or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP-inactivated enzyme. Co(NH3)4ATP inactivates Na+/K+-ATPase by binding to the low-affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)-ATP or Rh(III)-ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low-affinity ATP site. Inactivation of Na+/K+-ATPase was faster by the .DELTA. isomer of Co(NH3)4ATP than by the .delta. isomer. Co(NH3)4ATP, but not Cr(H20)4ATP or adenosine 5''-[.beta.,.gamma.-methylene]triphosphate competitively inhibited K+-activated p-nitrophenylphosphatase activity of Na+/K+-ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low-affinity ATP binding site.

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