84.Ki [mM]0.03160.001 0.8160.n1.960.1 2.060.Ks [mM]3.660.5 4.160.Kis [mM]3567b 0.6360.08 0.5760.kcat (s21)21.762.2 24.762.The dissociation constant of the enzyme-substrate complex (Ks), the inhibition constant of FBPase by its substrate (Kis) and b values were calculated assuming the model of partial noncompetitive inhibition by substrate [18]. The Hill equation was used to calculate dissociation constants for Mg2+, Ca2+ and AMP. Ki is a dissociation (inhibitory) constant for AMP or Ca2+, Ka is a dissociation (activatory) constant for Mg2+ and n is the Hill constant. The mean values and respective standard error calculated from 3 independent experiments are presented in the Table. doi:10.1371/journal.pone.0076669.tPLOS ONE | www.plosone.orgCa2+ Competes with Mg2+ for Binding to FBPaseFigure 2. Fluorescence spectra of the Tyr57Trp mutant under different ligation conditions. A) Enzyme under R-state conditions of ligation (5 mM F6P and 5 mM KPi) in the presence of various concentration of Ca2+ and Mg2+.Rapamycin B) Enzyme under R-state conditions of ligation (5 mM F6P and 5 mM KPi) in the presence of various concentration of Mg2+ and under T-state conditions of ligation (5 mM F6P, 5 mM KPi, and 2 mM AMP) in the presence of Mg2+.Evodiamine C) Enzyme under R-state conditions of ligation (5 mM F6P and 5 mM KPi) in the presence of various concentration of Zn2+ and under T-state conditions of ligation (5 mM F6P, 5 mM KPi, and 2 mM AMP) in the presence of Zn2+.PMID:24633055 The final emission spectra do not depend on the sequence of the ligands addition. doi:10.1371/journal.pone.0076669.gand KPi) were added to the enzyme in the absence of the activatory metal cations (data not shown). Both complexes, FBPase-activatory metal cations and FBPase-substrates, are inactive because loop 522 cannot adopt the engaged conformation, although the tetramer is in R-state. The addition of activatory metal cations to F6P- and KPisaturated FBPase caused an increase in the fluorescence intensity of Trp57 by about 115 and a red shift of lmax, from 348 nm to about 351 and 353 nm for Mg2+ and Zn2+, respectively (Fig. 2, Table 3). Evidently, these changes are correlated with the activation of the enzyme by divalent cations (Fig. 2, Table 2 3) and hence, with a conformational shift of loop 522 from its disengaged towards the engaged state. Addition of AMP or Ca2+ at concentrations completely inhibiting muscle FBPase to any of the FBPase-F6P-Pi-activatory cations complexes resulted in a decrease in fluorescence intensity and in a blue shift of lmax (Fig. 2 B , Table 3) reversing the changes induced by Mg2+ or Zn2+. In fact, the emission spectra of these FBPase complexes were nearly identical to those recorded in the absence of the activatory metal cations (Table 3). This indicates that the inactive, saturated with AMP or Ca2+, or depleted of the activatory cations FBPase adopts a disengaged-like conformation of loop 522.The Effect of Calcium on the Subcellular Localization of Various Forms of Muscle FBPaseSince it is known that Ca2+ concentrations that inhibit muscle FBPase also influence its interaction with its cellular binding partners [16,32], we tested the influence of elevated [Ca2+] on the localization of WT FBPase and the Tyr57Trp mutant in skeletal muscle fibers. TRITC-labeled WT muscle FBPase accumulated on the sarcomeric Z-lines (Fig. 3; [25]), as did the FITC-labeled Tyr57Trp muscle FBPase mutant. In the presence of 10 mM Ca2+, WT FBPase dissociated from the Z-line. In the same conditi.