Mechanism of Mg2+ 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 (Kd) 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 Kd of Mg2+-ATP interaction in solution. Within the enzyme-ATP-Mg2+ complex, the actual Kd 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.