Mutational analysis of Glu771 of the Ca2+‐ATPase of sarcoplasmic reticulum Effect of positive charge on dephosphorylation

FEBS Letters

Andersen, Jens Peter¹ 

[1] Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus, Ole Worms Allé 160, Universitetsparken, DK-8000 Aarhus C, Denmark

关键词: Calcium;    Proton countertransport;    Glutamate;    Lysine;    Mutant;    CrATP;    β;    γ-bidentate chromium(III) complex of ATP;    E2P;    phosphoenzyme intermediate with luminallly facing transport sites and ADP-insensitivity of the phosphoryl aspartyl bond;    EGTA;    [ethylenebis(oxyethylene-nitrilo)] tetraacetic acid;    HPLC;    high-performance liquid chromatography;    M1–M10;    putative transmembrane segments numbered from the NH2-terminal end of the peptide;    MOPS;    3-(N-morpholino)propanesulfonic acid;    TES;    N-tris[hydroxymethyl]-methyl-2-aminoethanesulfonic acid;    SDS;    sodium dodecyl sulfate;   

DOI  :  10.1016/0014-5793(94)01100-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown