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 1 -like configuration, allowing Na + but not K + to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector.