Mutational analysis of Glu771of the Ca2+-ATPase of sarcoplasmic reticulum Effect of positive charge on dephosphorylation

Abstract

The glutamic acid residue Glu⁷⁷¹ in the fifth transmembrane segment M5 of the Ca²⁺-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 Pi 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 Ca²⁺-ATPase cycle.