title: 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.
abstract: Purple acid phosphatases (PAPs) employ a dinuclear Fe(3+)Fe(2+) or Fe(3+)Zn(2+) 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 (K(i(comp)) approximately K(i(uncomp)) approximately 2 mM) to uncompetitive at pH 5.0 (K(i(uncomp)) = 0.2 mM). The inhibition constant for AlZn-BSPAP at pH 5.0 (K(i) = 3 microM) is approximately 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.PO(4).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.
authors.affiliation: E.C. Slater Institute, Biocentrum Amsterdam, University of Amsterdam, The Netherlands.
authors.forename: M W
authors.initials: MW
authors.lastname: Pinkse
authors.forename: M
authors.initials: M
authors.lastname: Merkx
authors.forename: B A
authors.initials: BA
authors.lastname: Averill
mesh_terms: D000135:Acid Phosphatase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005459:Fluorides / Q000737:chemistry*; D006023:Glycoproteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D008667:Metalloproteins / Q000037:antagonists & inhibitors; D008956:Models, Chemical; D010710:Phosphates / Q000737:chemistry*; D013154:Spleen; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D006023:Glycoproteins; D046911:Macromolecular Substances; D008667:Metalloproteins; D010710:Phosphates; D007501:Iron; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D015032:Zinc; D005459:Fluorides