Evidence for an Essential Role of a Conserved Threonine in the Conformational Change of P‐type Pumps

Ionic strength‐dependence of the conformational change in P‐type pumps indicates that electrostatic interactions contribute to the driving force for domain closure and stabilization of the E 2 conformation. The only conserved interaction predicted by a homology model of pig Na,K‐ATPase was a hydrogen bond between amino acids in the phosphorylation (T631) and nucleotide domains (G553) of the Ca‐ATPase template (1IWO). Therefore, the corresponding threonine in human Na,K‐ATPase (T623) was changed to serine or valine to test the hypothesis that the side‐chain hydroxyl group is important for the conformational rearrangement. The effect of the mutations on stable oxygen isotope exchange between P 18 O 4 and H 2 16 O was measured to diagnose for conformation (E 2 )‐dependent function. T623V did not catalyze 18 O exchange, thereby confirming an essential role without establishing the exact function. However, the conservative mutant T623S could be studied as a function of the K + and Na + as well as the Mg 2+ and P i concentrations. The data were fit to equations derived for ordered binding of Mg 2+ before P i and for a concerted change in conformation regulated by the transported ions. Opposing effects on the intrinsic Mg 2+ dissociation constant and the half‐maximum concentration for P i binding to the metalloenzyme and an increase in the half‐maximum K + concentration compared to wild‐type enzyme were estimated.