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.

ABSTRACT: 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 E₁-like configuration, allowing Na⁺ but not K⁺ to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector.

SUBMITTER: Nielsen HN 

PROVIDER: S-EPMC10812997 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature