Fluoride Inhibition of Bovine Spleen Purple Acid Phosphatase: Characterization of a Ternary Enzyme−Phosphate−Fluoride Complex as a Model for the Active Enzyme−Substrate−Hydroxide Complex

Purple acid phosphatases (PAPs) employ a dinuclear Fe3+Fe2+ or Fe3+Zn2+ center to catalyze the hydrolysis of phosphate monoesters. The interaction of fluoride with bovine spleen purple acid phosphatase (BSPAP) has been studied using a combination of steady-state kinetics and spectroscopic methods. For FeZn−BSPAP, the nature of the inhibition changes from noncompetitive at pH 6.5 (Ki(comp) ≈ Ki(uncomp) ≈ 2 mM) to uncompetitive at pH 5.0 (Ki(uncomp) = 0.2 mM). The inhibition constant for AlZn−BSPAP at pH 5.0 (Ki = 3 μM) is ∼50−70-fold lower than that observed for both FeZn−BSAP and GaZn−BSPAP, suggesting that fluoride binds to the trivalent metal. Fluoride binding to the enzyme−substrate complex was found to be remarkably slow; hence, the kinetics of fluoride binding were studied in some detail for FeZn−, AlZn−, and FeFe−BSPAP at pH 5.0 and for FeZn−BSPAP at pH 6.5. Since the enzyme kinetics studies indicated the formation of a ternary enzyme−substrate-fluoride complex, the binding of fluoride to FeZn−BSPAP was studied using optical and EPR spectroscopies, both in the presence and absence of phosphate. The characteristic optical and EPR spectra of FeZn−BSPAP·F and FeZn−BSPAP·PO4·F are similar at pH 5.0 and pH 6.5, indicating the formation of similar fluoride complexes at both pHs. A structural model for the ternary enzyme−(substrate/phosphate)-fluoride complexes is proposed that can explain the results from both the spectroscopic and the enzyme kinetics experiments. In this model, fluoride binds to the trivalent metal replacing the water/hydroxide ligand that is essential for the hydrolysis reaction to take place, while phosphate or the phosphate ester coordinates to the divalent metal ion.