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 been purified to homogeneity and characterized using spectroscopic investigations. Matrix-assisted laser desorption/ionization mass spectrometry analysis revealed a molecular mass of approximately 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 pKa 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.
Durmus
A
A
Anorganisch-Chemisches Institut, Homburg, Germany.
Eicken
C
C
Sift
B H
BH
Kratel
A
A
Kappl
R
R
Hüttermann
J
J
Krebs
B
B
D000135:Acid Phosphatase / Q000737:chemistry* / Q000502:physiology; D000595:Amino Acid Sequence; D001665:Binding Sites; D006023:Glycoproteins / Q000737:chemistry* / Q000502:physiology; D007501:Iron / Q000032:analysis; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D019657:Solanaceae / Q000201:enzymology*; D013052:Spectrometry, X-Ray Emission; D015032:Zinc / Q000032:analysis
D006023:Glycoproteins; D007501:Iron; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D015032:Zinc