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 Martijn W. H. Pinkse, Maarten Merkx, and Bruce A. Averill* E.C. Slater Institute, Biocentrum Amsterdam, UniVersity of Amsterdam, Plantage Muidergracht 12, 1018 TV, Amsterdam, The Netherlands ReceiVed February 24, 1999; ReVised Manuscript ReceiVed June 7, 1999 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) ≈ K i(uncomp) ≈ 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 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‚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. Purple acid phosphatases (PAPs) 1 belong to the growing group of metalloenzymes that employ a di- or trinuclear metal center to catalyze hydrolysis reactions (1, 2). All mammalian PAPs contain a diiron center, which is catalytically active in the mixed-valent Fe(III)-Fe(II) oxidation state (3). Two of those have been the subject of extensive spectroscopic and enzyme kinetics studies, uteroferrin (isolated from the uteral fluids of pregnant pigs; Uf) and bovine spleen purple acid phosphatase (BSPAP). The plant PAP isolated from red kidney beans (KBPAP) is the only PAP for which an X-ray structure has been determined (4, 5). This PAP contains an Fe(III)Zn(II) metal center, but shows kinetics and spectro- scopic properties similar to those of the mammalian enzymes. All amino acids found to coordinate the metals in KBPAP are strictly conserved among all PAP sequences, even those from microorganisms such as Aspergillus ficum (3, 6). Moreover, a sequence motif that incorporates most of these amino acids is found in a much larger group of phospho- hydrolases, among which are exonucleases, 5′-nucleotidases, diadenosinetetraphosphatases, and Ser/Thr specific protein phosphatases (5, 7, 8). X-ray structure determinations of two Ser/Thr specific protein phosphatases, PP1 and PP2B (cal- cineurin), have revealed a dinuclear metal center resembling that of the PAPs, except for the tyrosinate ligand to the Fe 3+ that gives the PAPs their characteristic purple color. The PAPs have a number of properties that permit the molecular details by which these dinuclear metal centers participate in the hydrolysis of phosphate esters to be studied: (1) they have favorable spectroscopic properties (intense charge- transfer band, characteristic EPR signals in the active Fe(III)- Fe(II) and Fe(III)Zn(II) oxidation states, Mo ¨ssbauer active metal ions) that provide probes to follow processes taking place in the coordination environment of the metal center; (2) unlike the PPs, the PAPs are relatively stable enzymes whose activity is not regulated via complicated activation mechanisms; (3) each of the two metals can be specifically * To whom correspondence should be addressed. Telephone: 31- 20-5255045. Fax: 31-20-5255124. E-mail: BAA@chem.uva.nl. 1 Abbreviations: AlZn-BSPAP, BSPAP with aluminum at the ferric site and zinc at the ferrous site; BSPAP, bovine spleen purple acid phosphatase; EPR, electron paramagnetic resonance; EXAFS, extended X-ray absorption fine structure; FeFe-BSPAP, BSPAP with iron at the ferric and ferrous sites; FeZn-BSPAP, BSPAP with iron at the ferric site and zinc at the ferrous site; FeZn-BSPAP‚F, FeZn-BSPAP complexed with fluoride; FeZn-BSPAP‚PO 4, FeZn-BSPAP com- plexed with phosphate; FeZn-BSPAP‚PO4‚F, FeZn-BSPAP com- plexed with phosphate and fluoride; FeZn-Uf, uteroferrin with iron at the ferric site and zinc at the ferrous site; GaZn-BSPAP, BSPAP with gallium at the ferric site and zinc at the ferrous site; KBPAP, purple acid phosphatase of red kidney beans; MES, 2-[N-morpholino]- ethanesulfonic acid; PAP, purple acid phosphatase; p-NPP, disodium salt of p-nitrophenyl phosphate; PP, protein phosphatase; PP1, protein phosphatase 1; PP2B, protein phosphatase 2B; Uf, uteroferrin. 9926 Biochemistry 1999, 38, 9926-9936 10.1021/bi990446w CCC: $18.00 © 1999 American Chemical Society Published on Web 07/20/1999