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

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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 Pi, 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.