With the aim of critically assessing the current models for metal ion assisted hydrolytic reaction mechanisms, an updated review of the current understanding of metallohydrolase-catalyzed reactions is presented. Focus is on four systems, purple acid phosphatases (PAPs), Ser/Thr protein phosphatases (PPs), 3′-5′ exonucleases, and 5′-nucleotidases (5′-NTs), which have contributed to major advancement of the understanding of the catalytic mechanisms that operate in such enzymes.

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The catalytic mechanisms of binuclear metallohydrolases.

https://espace.library.uq.edu.au/view/UQ:59608/UQ59608_OA.pdf

A Purple Acid Phosphatase from Sweet Potato Contains an Antiferromagnetically Coupled Binuclear Fe-Mn Center

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.

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

Magnetostructural correlations in exchange coupled phenoxo-, alkoxo-, and hydroxo-bridged dinuclear iron(III) compounds

Purple acid phosphatases (PAPs) contain a dinuclear metal center in their active site and hydrolyze phosphoric acid esters at low pH. Characteristic of this group of acid phosphatases is their resistance to inhibition by tartrate and their purple color, due to the presence of a tyrosine residue ligated to a ferric iron. The mammalian enzymes all contain a mixed-valent di-iron unit in their catalytic active form, first identified in the bovine spleen and porcine uterus enzymes, while a heterodinuclear Fe(III)Zn(II) unit has been characterized for the most studied plant enzyme from kidney bean. The enzymes from porcine uterus and bovine spleen can be converted into active FeZn forms and the plant enzyme can be transformed into an active FeFe form. In recent years the dimetal center of PAPs has been studied using numerous spectroscopic methods such as Mossbauer spectroscopy, EPR, NMR, EXAFS, magnetic, electrochemical and resonance Raman studies characterizing most of the metal coordinating residues, the metal-metal separation and providing evidence of the similarity between enzymes from different sources. Analysis of the products of hydrolysis of a substrate containing a chiral phosphorus by 31P NMR, stopped-flow measurements and kinetic studies all support a reaction path involving nucleophilic attack of a Fe(III)-bound hydroxide ligand on the phosphate ester. The recently solved crystal structure of the plant enzyme provides the structural basis for the understanding of the two-metal ion mechanism of this class of enzymes.

R. Löcke

Papers

Magnetic susceptibility studies on the diiron forms of the metalloprotein purple acid phosphate from bovine spleen and kidney bean