Pinkse M W, Merkx M, Averill B A
E.C. Slater Institute, Biocentrum Amsterdam, University of Amsterdam, The Netherlands.
Biochemistry. 1999 Aug 3;38(31):9926-36. doi: 10.1021/bi990446w.
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.
紫色酸性磷酸酶（PAPs）利用双核Fe(3+)Fe(2+)或Fe(3+)Zn(2+)中心催化磷酸单酯的水解。已结合稳态动力学和光谱学方法研究了氟化物与牛脾紫色酸性磷酸酶（BSPAP）的相互作用。对于FeZn-BSPAP，抑制性质从pH 6.5时的非竞争性（K(i(comp))≈K(i(uncomp))≈2 mM）转变为pH 5.0时的反竞争性（K(i(uncomp)) = 0.2 mM）。AlZn-BSPAP在pH 5.0时的抑制常数（K(i) = 3 μM）比FeZn-BSAP和GaZn-BSPAP的抑制常数低约50 - 70倍，这表明氟化物与三价金属结合。发现氟化物与酶 - 底物复合物的结合非常缓慢；因此，详细研究了pH 5.0时FeZn -、AlZn -和FeFe - BSPAP以及pH 6.5时FeZn - BSPAP的氟化物结合动力学。由于酶动力学研究表明形成了三元酶 - 底物 - 氟化物复合物，因此在有和没有磷酸盐存在的情况下，使用光学和电子顺磁共振光谱研究了氟化物与FeZn - BSPAP的结合。FeZn - BSPAP.F和FeZn - BSPAP.PO(4).F在pH 5.0和pH 6.5时的特征光学和电子顺磁共振光谱相似，表明在这两个pH值下形成了相似的氟化物复合物。提出了三元酶 -（底物/磷酸盐）- 氟化物复合物的结构模型，该模型可以解释光谱学和酶动力学实验的结果。在该模型中，氟化物与三价金属结合，取代了水解反应发生所必需的水/氢氧根配体，而磷酸盐或磷酸酯与二价金属离子配位。
