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+ cen ter to catalyze the hydrolysis of phosphate monoesters. The interaction of fluoride with bovine spleen purple acid phosphatase (BSPAP) has been studie d using a combination of steady-state kinetics and spectroscopic methods. F or FeZn-BSPAP, the nature of the inhibition changes from noncompetitive at pH 6.5 (K-i(comp) approximate to K-i(uncomp) approximate to 2 mM) to uncomp etitive at pH 5.0 (K-i(uncomp) = 0.2 mM). The inhibition constant for AlZn- BSPAP at pH 5.0 (K-i = 3 mu M) is similar to 50-70-fold lower than that obs erved for both FeZn-BSAP and GaZn-BSPAP, suggesting that fluoride binds to the trivalent metal. Fluoride binding to the enzyme-substrate complex was f ound to be remarkably slow; hence, the kinetics of fluoride binding were st udied in some detail for FeZn-, AlZn-, and FeFe-BSPAP at pH 5.0 and for FeZ n-BSPAP at pH 6.5. Since the enzyme kinetics studies indicated the formatio n of a ternary enzyme-substrate-fluoride complex, the binding of fluoride t o FeZn-BSPAP was studied using optical and EPR spectroscopies, both in the presence and absence of phosphate. The characteristic optical and EPR spect ra 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 str uctural model for the ternary enzyme-(substrate/phosphate)-fluoride complex es is proposed that can explain the results from both the spectroscopic and the enzyme kinetics experiments. In this model, fluoride binds to the triv alent metal replacing the water/hydroxide ligand that is essential for the hydrolysis reaction to take place, while phosphate or the phosphate ester c oordinates to the divalent metal ion.