Mutational analysis of the role of Glu309 in the sarcoplasmic reticulum Ca2+‐ATPase of frog skeletal muscle

FEBS Letters

Andersen, Jens Peter¹  Vilsen, Bente¹ 

[1] Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark

关键词: Ca2+-ATPase;    Sarcoplasmic reticulum;    Amino acid substitution;    Ca2+ binding;    Phosphoenzyme intermediate;    Ca2+-ATPase;    Ca2+-activated adenosine triphosphatase;    E1P;    ADP-sensitive phosphoenzyme intermediate;    E2P;    ADP-insensitive phosphoenzyme intermediate;    EGTA;    [ethylenebis(oxy-ethylene-nitrilo)tetraacetic acid;    MES;    2-(N-morpholino)-ethanesulfonic acid;    MOPS;    3-(N-morpholino)propanesulfonic acid;    TES;    N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid;    SDS;    sodium dodecyl sulfate;   

DOI  :  10.1016/0014-5793(92)81010-J

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Site-specific mutagenesis was used to analyse the role of the residue, Glu³⁰⁹, in the function of the Ca²⁺-ATPase of frog skeletal muscle sarcoplasmic reticulum by substitution with Ala or Lys. At pH 6.0, 100 μM Ca²⁺ was unable to prevent phosphorylation from P_i, consistent with previous observations on the Ca²⁺-ATPase of rabbit fast twitch muscle [Clarke, D.M., Loo, T.W, Inesi, G. and MacLennan, D.H. (1989) Nature 339, 476–478]. At neutral pH, however, micromolar concentrations of Ca²⁺ were sufficient to inhibit phosphorylation of the Glu³⁰⁹→Lys mutant from inorganic phosphate, suggesting that at least one high-affinity Ca²⁺ site was relatively intact in this mutant. The Glu³⁰⁹→Lys mutant was unable to form a phosphoenzyme from ATP at all Ca²⁺ concentrations studied (up to 12.5 mM), whereas phosphorylation of the Glu³⁰⁹→Ala mutant occurred at 12.5 mM Ca²⁺, but not at Ca²⁺ concentrations in the submillimolar range. Kinetic studies demonstrated a reduced rate of dephosphorylation of the E₂P intermediate in the Glu³⁰⁹→Lys mutant. A less pronounced stabilization of E₂P was observed with the Glu³⁰⁹→Ala mutant, suggesting a possible role of the charge at the position of Glu³⁰⁹ in phosphoenzyme hydrolysis.

【 授权许可】

Unknown