The active site of purple acid phosphatase from sweet potatoes (Ipomoea batatas) -: Metal content and spectroscopic characterization
The active site of purple acid phosphatase from sweet potatoes (Ipomoea batatas) -: Metal content and spectroscopic characterization
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DOI：
10.1046/j.1432-1327.1999.00230.x
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发表时间：
1999-03-01
期刊：
影响因子：
--
通讯作者：
Krebs, B
中科院分区：
文献类型：
--
作者：
Durmus, A;Eicken, C;Krebs, B
甘薯紫色酸性磷酸酶（spPAP）已被纯化至均一，并使用光谱研究其特征。基质辅助激光解吸/电离质谱分析显示分子量约为112 kDa。使用同步辐射通过X射线荧光测定金属含量。与以前的研究相反，它表明，spPAP包含一个铁（III）-锌（II）中心的活性位点，如以前确定的紫色酸性磷酸酶从红芸豆（KbPAP）。此外，氨基酸序列的比对表明，参与金属结合的残基在两种植物PAP中相同。酪氨酸作为发色团Fe（III）的配体之一发挥作用。spPAP的低温EPR谱显示g = 4.3附近的信号，这是菱形环境中高自旋Fe（III）的特征。的Tyr-Fe（III）的电荷转移跃迁和EPR信号都是非常敏感的pH值的变化。pH值的依赖性强烈表明存在的pK（a）为4.7的可电离的基团，所产生的水配体配位到Fe（III）。EPR和UV/可见光研究的spPAP在存在的抑制剂磷酸盐或砷酸盐表明，这两种阴离子结合到Fe（III）的双核中心取代协调的水或氢氧化物的水解所需的配体。与kbPAP中的His 202和His 296相对应的spPAP保守的组氨酸残基可能在催化中相互作用。
Purple acid phosphatase from sweet potatoes Ipomoea batatas (spPAP) has been purified to homogeneity and characterized using spectroscopic investigations. Matrix-assisted laser desorption/ionization mass spectrometry analysis revealed a molecular mass of approximate to 112 kDa. The metal content was determined by X-ray fluorescence using synchrotron radiation. In contrast to previous studies it is shown that spPAP contains a Fe(III)-Zn(II) center in the active site as previously determined for the purple acid phosphatase from red kidney bean (kbPAP). Moreover, an alignment of the amino acid sequences suggests that the residues involved in metal-binding are identical in both plant PAPs. Tyrosine functions as one of the ligands for the chromophoric Fe(III). Low temperature EPR spectra of spPAP show a signal near g = 4.3, characteristic for high-spin Fe(III) in a rhombic environment. The Tyr-Fe(III) charge transfer transition and the EPR signal are both very sensitive to changes in pH. The pH dependency strongly suggests the presence of an ionizable group with a pK(a) of 4.7, arising from an aquo ligand coordinated to Fe(III). EPR and UV/visible studies of spPAP in the presence of the inhibitors phosphate or arsenate suggest that both anions bind to Fe(III) in the binuclear center replacing the coordinated water or hydroxide ligand necessary for hydrolysis. The conserved histidine residues of spPAP corresponding to His202 and His296 in kbPAP probably interact in catalysis.