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 reticu lum was substituted with lysine, alanine, and glycine by site-directed mutagenesis. Mutant Glu(771)-->Lys was unable to occlude Ca2+, and Ca 2+ did not inhibit phosphorylation from P-i or activate phosphorylatio n from ATP of this mutant. Mutants Glu(771)-->Ala and Glu(771)-->Gly w ere likewise unable to occlude Ca2+, but Ca2+ in the millimolar concen tration range activated phosphorylation from ATP and inhibited phospho rylation from P-i of these mutants. The dephosphorylation of the ADP-i nsensitive E2P phosphoenzyme intermediate of mutants Glu(771)-->Ala an d Glu(771)-->Gly was found to be blocked, whereas the dephosphorylatio n proceeded rapidly for mutant Glu(771)-->Lys, This finding suggests a role of the positive charge of the lysine in induction of dephosphory lation, supporting the hypothesis that the side chain of Glu(771) part icipates in the countertransport of two protons per Ca2+-ATPase cycle.