MUTATIONAL ANALYSIS OF GLU(771) OF THE CA2+-ATPASE OF SARCOPLASMIC-RETICULUM - EFFECT OF POSITIVE CHARGE ON DEPHOSPHORYLATION

The glutamic acid residue Glu(771) 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 Glu(771)-->Lys was unable to occlude Ca2+, and Ca2+ did not inhibit phosphorylation from P-i or activate phosphorylation from ATP of this mutant. Mutants Glu(771)-->Ala and Glu(771)-->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 Glu(771)-->Ala and Glu(771)-->Gly was found to be blocked, whereas the dephosphorylation proceeded rapidly for mutant Glu(771)-->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 Glu(771) participates in the countertransport of two protons per Ca2+-ATPase cycle.