The active site of purple acid phosphatase from sweet potatoes (Ipomoea batatas) - Metal content and spectroscopic characterization

Purple acid phosphatase from sweet potatoes Ipomoea batatas (spPAP) has bee n purified to homogeneity and characterized using spectroscopic investigati ons. 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 phosphatas e from red kidney bean (kbPAP). Moreover, an alignment of the amino acid se quences suggests that the residues involved in metal-binding are identical in both plant PAPs. Tyrosine functions as one of the ligands for the chromo phoric 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 Ty r-Fe(III) charge transfer transition and the EPR signal are both very sensi tive 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 coordin ated 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 corres ponding to His202 and His296 in kbPAP probably interact in catalysis.