Mechanism of Mg Binding in the Na,K-ATPase

The Mg²⁺ dependence of the kinetics of the phosphorylation and conformational changes of Na⁺,K⁺-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421. The enzyme was preequilibrated in buffer containing 130 mM NaCl to stabilize the E1(Na⁺)₃ state. On mixing with ATP, a fluorescence increase was observed. Two exponential functions were necessary to fit the data. Both phases displayed an increase in their observed rate constants with increasing Mg²⁺ to saturating values of 195 (± 6) s⁻¹ and 54 (± 8) s⁻¹ for the fast and slow phases, respectively. The fast phase was attributed to enzyme conversion into the E2MgP state. The slow phase was attributed to relaxation of the dephosphorylation/rephosphorylation (by ATP) equilibrium and the buildup of some enzyme in the E2Mg state. Taking into account competition from free ATP, the dissociation constant (K_d) of Mg²⁺ interaction with the E1ATP(Na⁺)₃ state was estimated as 0.069 (± 0.010) mM. This is virtually identical to the estimated value of the K_d of Mg²⁺-ATP interaction in solution. Within the enzyme-ATP-Mg²⁺ complex, the actual K_d for Mg²⁺ binding can be attributed primarily to complexation by ATP itself, with no apparent contribution from coordination by residues of the enzyme environment in the E1 conformation.