Mutational analysis of Glu771 of the Ca(2+)-ATPase of sarcoplasmic reticulum. Effect of positive charge on dephosphorylation.

The glutamic acid residue Glu771 in the fifth transmembrane segment M5 of the Ca(2+)-ATPase of rabbit fast twitch muscle sarcoplasmic reticulum was substituted with lysine, alanine, and glycine by site-directed mutagenesis. Mutant Glu771-->Lys was unable to occlude Ca2+, and Ca2+ did not inhibit phosphorylation from P(i) or activate phosphorylation from ATP of this mutant. Mutants Glu771-->Ala and Glu771-->Gly were likewise unable to occlude Ca2+, but Ca2+ in the millimolar concentration range activated phosphorylation from ATP and inhibited phosphorylation from P(i) of these mutants. The dephosphorylation of the ADP-insensitive E2P phosphoenzyme intermediate of mutants Glu771-->Ala and Glu771-->Gly was found to be blocked, whereas the dephosphorylation proceeded rapidly for mutant Glu771-->Lys. This finding suggests a role of the positive charge of the lysine in induction of dephosphorylation, supporting the hypothesis that the side chain of Glu771 participates in the countertransport of two protons per Ca(2+)-ATPase cycle.