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 < 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.Keywords: EPR; Ipomoea batatas; metalloenzyme; purple acid phosphatase; X-ray fluorescence.Purple acid phosphatases (PAPs) with a Fe(III)±Me(II) center in their active site catalyze the hydrolysis of activated phosphoric acid esters and phosphoric acid anhydrides in the pH range 4±7. These metalloenzymes are resistant to inhibition by tartrate and show a characteristic Tyr±Fe(III) charge transfer (CT) band at <560 nm responsible for the characteristic purple color of the enzyme (for review see Refs 1±4).Mammalian PAPs or tartrate-resistant acid phosphatases (TRAPs) are monomeric glycoproteins with a molecular mass of <35 kDa containing a Fe(III)±Fe(II) unit in the active site. Recently it has been shown that the residual activity of oxidized bovine spleen purple acid phosphatase (bsPAP) originates from an`impurity' of bsPAP with iron at the ferric site and zinc at the ferrous site (FeZn-bsPAP) in the native form [5].For mammalian acid phosphatases multiple physiological functions, i.e. the degradation of aged erythrocytes [6,7] and active bone resorption [8,9] have been discussed. Although no crystal structure is available for any of these enzymes at present, the dimetal system has been characterized by several spectroscopic methods including Mo Èûbauer spectroscopy [5,10±13] A breakthrough was achieved with the determination of the three-dimensional structure of kbPAP [40,41]. Based on this crystal structure and the amino acid sequence alignment, it has been shown that the predicted secondary structure of the mammalian PAP uteroferrin (Uf) is similar in foldin...
XAS investigations on the active site of the purple acid phosphatase isolated from the sweet potato Ipomoea batatas provided first information about the structure. Data analysis reveal a dinuclear active site containing an iron and a zinc atom. The first coordination sphere of each metal in the active site consists of six nitrogen/oxygen donor atoms, resulting in a distorted octahedral coordination sphere very similar to the active site of the enzyme isolated from the red kidney bean Phaseolus vulgaris. The XAS spectra were recorded in fluorescence mode at 18 K using a Canberra 13-element germanium solid state detector.
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