Na,K-ATPase with Disrupted Na Binding Sites I and III Binds Na with Increased Affinity at Site II and Undergoes Na-Activated Phosphorylation with ATP.

Na,K-ATPase actively extrudes three cytoplasmic Na ions in exchange for two extracellular K ions for each ATP hydrolyzed. The atomic structure with bound Na identifies three Na sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na affinity. This indicates that Na binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E-like configuration, allowing Na but not K to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector.