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

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Abstract
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 ÌM) 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.