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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- 作者：Martijn W. H. Pinkse ; Maarten Merkx ; and Bruce A. Averill
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：August 3, 1999
- 年：1999
- 卷：38
- 期：31
- 页码：9926 - 9936
- 全文大小：148K
- 年卷期：v.38,no.31(August 3, 1999)
- ISSN：1520-4995
文摘
Purple acid phosphatases (PAPs) employ a dinuclear Fe3+Fe2+ or Fe3+Zn2+ center to catalyzethe hydrolysis of phosphate monoesters. The interaction of fluoride with bovine spleen purple acidphosphatase (BSPAP) has been studied using a combination of steady-state kinetics and spectroscopicmethods. 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 
M) is ~50-70-fold lower than that observed for both FeZn-BSAP andGaZn-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 studiedin some detail for FeZn-, AlZn-, and FeFe-BSPAP at pH 5.0 and for FeZn-BSPAP at pH 6.5. Sincethe enzyme kinetics studies indicated the formation of a ternary enzyme-substrate-fluoride complex, thebinding of fluoride to FeZn-BSPAP was studied using optical and EPR spectroscopies, both in the presenceand 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 complexesat both pHs. A structural model for the ternary enzyme-(substrate/phosphate)-fluoride complexes isproposed 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 essentialfor the hydrolysis reaction to take place, while phosphate or the phosphate ester coordinates to the divalentmetal ion.
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 M) is ~50-70-fold lower than that observed for both FeZn-BSAP andGaZn-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 studiedin some detail for FeZn-, AlZn-, and FeFe-BSPAP at pH 5.0 and for FeZn-BSPAP at pH 6.5. Sincethe enzyme kinetics studies indicated the formation of a ternary enzyme-substrate-fluoride complex, thebinding of fluoride to FeZn-BSPAP was studied using optical and EPR spectroscopies, both in the presenceand 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 complexesat both pHs. A structural model for the ternary enzyme-(substrate/phosphate)-fluoride complexes isproposed 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 essentialfor the hydrolysis reaction to take place, while phosphate or the phosphate ester coordinates to the divalentmetal ion.