Mutational analysis of the role of Glu309in the sarcoplasmic reticulum Ca2+-ATPase of frog skeletal muscle

Abstract

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.