Mutational effects on conformational changes of the dephospho- and phospho-forms of the Na+,K+-ATPase

Gly263 of the rat kidney Na+,K+-ATPase is highly conserved within the famil y of P-type ATPases. Mutants in which Gly263 or the juxtaposed Arg264 had b een replaced by alanine were expressed at high levels in COS-1 cells and ch aracterized functionally. Titrations of Na+,K+, ATP, and vanadate dependenc ies of Na+,K+-ATPase activity showed changes in the apparent affinities rel ative to wild-type compatible with a displacement of the E-1-E-2 conformati onal equilibrium in favor of E-1. The level of the K+-occluded form was red uced in the Gly263 --> Ala and Arg264 --> Ala mutants, and the rate constan t characterizing deocclusion of K+ or Rb+ was increased as much as 20-fold in the Gly263 --> Ala mutant. Studies of the sensitivity of the phosphoenzy me to K+ and ADP showed a displacement of the E1P-E2P equilibrium of the ph osphoenzyme in favor of E1P, and dephosphorylation experiments carried out at 25 degreesC on a millisecond time scale using a quenched-flow technique demonstrated a reduction of the E1P to E2P conversion rate in the mutants. Hence, the mutations displaced the conformational equilibria of dephosphoen zyme and phosphoenzyme in parallel in favor of the E-1 and E1P forms. The o bserved effects were more pronounced in the Gly263 --> Ala mutant compared with the Arg264 --> Ala mutant. Leu332 mutations that likewise displaced th e conformational equilibria in favor of E-1 and E1P were also studied. Unli ke the Gly263 --> Ala mutant the Leu332 mutants displayed a wild-type like rate of K+ deocclusion. Thus, the effect of the Gly263 mutation on the E-1- E-2 conformational equilibrium seems to be caused mainly by an acceleration of the K+-deoccluding step, whereas in the Leu332 mutants the rate of the reverse reaction seems to be reduced.