Mutational analysis of Glu771of the Ca2+-ATPase of sarcoplasmic reticulum Effect of positive charge on dephosphorylation
- 18 October 1994
- journal article
- Published by Wiley in FEBS Letters
- Vol. 354 (1) , 93-96
- https://doi.org/10.1016/0014-5793(94)01100-1
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 Pi 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.Keywords
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