Binding of fluoride by yeast enolase.
The kinetics of fluoride binding by yeast enolase have been examined by direct measurement of equilibrium fluoride (F) concentrations with an ion-specific electrode. Mg2+ and inorganic phosphate (Pi) affect the binding of F, and evidence is presented for an ordered binding mechanism in which phosphate and F interact with conformational and catalytic Mg2+ species, with the overall formation of a quaternary complex. A maximum of four atoms of F were bound per enzyme dimer, and the data point to the nonequivalent binding of pairs of F atoms. The dissociation constants were 5.0 X 10(-4) M and 8.2 X 10(-5) M for the first and second pairs of F atoms, respectively. The first pair of binding sites was filled when the ratio of phosphate ions/enolase dimer exceeded 2 and appeared to involve the pair of conformation Mg2+ ions. The binding of the second pair of F atoms followed the binding of catalytic Mg2+ in the presence of Pi and, furthermore, appeared to exhibit positive cooperativity with respect to F. The data suggest, also, that the binding of Pi may involve sequential addition of Pi pairs to the different Mg2+ species on the enzyme. F binding was at a maximum between pH 5.5 and pH 6.0, consistent with an involvement of the monovalent form of Pi. In the absence of added Pi, (MgF)+ appeared to be the preferred ligand. Addition of the enzyme substrate 2-phosphoglycerate led to the release of bound F. These findings are consistent with the known patterns of inhibition of enzymatic activity by F.
D004305:Dose-Response Relationship, Drug; D004566:Electrodes; D005459:Fluorides / Q000378:metabolism*; D005988:Glyceric Acids / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008278:Magnesium Sulfate / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013291:Streptococcus / Q000201:enzymology
D005988:Glyceric Acids; D010710:Phosphates; C008885:2-phosphoglycerate; D008278:Magnesium Sulfate; D010751:Phosphopyruvate Hydratase; D005459:Fluorides