Importance of conserved Thr214 in domain A of the Na+,K+-ATPase for stabilization of the phosphoryl transition state complex in E2P dephosphorylation

Abstract

Thr214 of the highly conserved 214TGES sequence in domain A of the Na+,K+-ATPase was replaced with alanine, and the mutant was compared functionally with the previously characterized domain A mutant Gly263 → Ala. Thr214 → Ala displayed a conspicuous 150-fold reduction of the apparent vanadate affinity for inhibition of ATPase activity, which could not simply be explained by the observed shifts of the conformational equilibria in favor of E1 and E1P. The intrinsic vanadate affinity of the E2 form and the effect on the apparent vanadate affinity of displacement of the E1-E2 equilibrium were determined in a phosphorylation assay that allows the enzyme-vanadate complex to be formed under equilibrium conditions. When the E2 form prevailed, Thr214 → Ala retained a reduced vanadate affinity relative to wild type, whereas the affinity of Gly263 → Ala became wild type-like. Thus, mutation of Thr214 affected the intrinsic affinity of E2 for vanadate. Furthermore, Thr214 → Ala showed at least a 5-fold reduced E2P dephosphorylation rate relative to wild type in the presence of saturating concentrations of K+ and Mg2+. Because vanadate is a phosphoryl transition state analog, it is proposed that defective binding of the phosphoryl transition state complex (transition state destabilization) causes the inability to catalyze E2P dephosphorylation properly. By contrast, the phosphorylation site in the E1 form was unaffected in Thr214 → Ala. Replacement of the glutamate, Glu216, of 214TGES with alanine was incompatible with cell viability, indicating a very low transport activity or expression level. Our results support the hypothesis that domain A is isolated in the E1 form, but contributes to make up the catalytic site in the E2 and E2P conformations.