Importance of Glu282 in transmembrane segment M3 of the Na+,K+-ATPase for control of cation interaction and conformational changes

Abstract

Glu282 located in the NH2-terminal part of transmembrane helix M3 of the Na+,K+-ATPase was replaced by alanine, glycine, leucine, lysine, aspartate, or glutamine, and the effects of the mutations on the overall and partial reactions of the enzyme were analyzed. The mutations affected at least 3 important functions of the Na+,K+-ATPase: (i) the conformational transitions between E1 and E2 forms of dephospho- and phosphoenzyme, (ii) Na+ binding at the cytoplasmically facing sites of E1, and (iii) long-range interaction controlling dephosphorylation. In mutants Glu282 → Lys and Glu282 → Asp, the E1 form was favored during ATP hydrolysis, whereas the E2 form was favored in Glu282 → Ala and Glu282 → Gly. Regardless of the change of conformational equilibrium, all the mutants displayed a reduced apparent affinity for Na+, at least Mold for Glu282 → Lys and Glu282 → Asp, suggesting a direct effect on the Na+ binding properties of E1. Glu282 → Ala and Glu282 → Gly exhibited an extraordinary high rate of ATP hydrolysis in the mere presence of Na+ without K+ ("Na+-ATPase activity"), because of an increased rate of dephosphorylation of E2P. These results are in accordance with the hypothesis that Glu282 is involved in the communication between the cation binding pocket and the catalytic site and in control of the cytoplasmic entry pathway for Na+.