Nucleotide Binding to Na,K-ATPase: The Role of Electrostatic Interactions

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Abstract
The contribution of electrostatic forces to the interaction of Na,K-ATPase with adenine nucleotides was investigated by studying the effect of ionic strength on nucleotide binding. At pH 7.0 and 20 °C, there was a qualitative correlation between the equilibrium dissociation constant (Kd) values for ATP, ADP, and MgADP and their total charges. All Kd values increased with increasing ionic strength. According to the Debye−Hückel theory, this suggests that the nucleotide binding site and its ligands have “effective” charges of opposite signs. However, quantitative analysis of the dependence on ionic strength shows that the product of the effective electrostatic charges on the ligand and the binding site is the same for all nucleotides, and is therefore independent of the total charge of the nucleotide. The data suggest that association of nucleotides with Na,K-ATPase is governed by a partial charge rather than the total charge of the nucleotide. This charge, interacting with positive charges on the protein, is probably the one corresponding to the α-phosphate of the nucleotide. Dissociation rate constants measured in complementary transient kinetic experiments were 13 s-1 for ATP and 27 s-1 for ADP, independent of the ionic strength in the range 0.1−0.5 M. This implies similar association rate constants for the two nucleotides (about 40 × 106 M-1 s-1 at I = 0.1 M). The results suggest that long-range Coulombic forces, affecting association rates, are not the main contributors to the observed differences in affinities, and that local interactions, affecting dissociation rates, may play an even greater role.