Electronic Structure and Spectro-Structural Correlations of Fe(III)Zn(II) Biomimetics for Purple Acid Phosphatases: Relevance to DNA Cleavage and Cytotoxic Activity

Purple acid phosphatases (PAPs) are a group of metallohydrolases that contain a dinuclear Fe(II)M(II) center (M(II) = Fe, Mn, Zn) in the active site and are able to catalyze the hydrolysis of a variety of phosphoric acid esters. The dinuclear complex [(H(2)O)Fe(III)(mu-OH)Zn(II)(L-H)](CIO(4))(2) (2) with the ligand 2-[N-bis(2-pyridylmethyl)aminomethyl]-4-methyl-6-[N-(2-pyridylmethyl)(2-hydroxybenzyl) aminomethyl]phenol (H(2)L-H) has recently been prepared and is found to closely mimic the coordination environment of the Fe(III)Zn(II) active site found in red kidney bean PAP (Neves et al. J. Am. Chem. Soc. 2007, 129, 7486). The biomimetic shows significant catalytic activity in hydrolytic reactions. By using a variety of structural, spectroscopic, and computational techniques the electronic structure of the Fe(III) center of this biomimetic complex was determined. In the solid state the electronic ground state reflects the rhombically distorted Fe(III)N(2)O(4) octahedron with a dominant tetragonal compression align ad along the mu-OH-Fe-O(phenolate) direction. To probe the role of the Fe-O(phenolate) bond, the phenolate moiety was modified to contain electron-donating or -withdrawing groups (-CH(3), -H, -Br, -NO(2)) in the 5-position. Tie effects of the substituents on the electronic properties of the biomimetic complexes were studied with a range of experimental and computational techniques. This study establishes benchmarks against accurate crystallographic struck ral information using spectroscopic techniques that are not restricted to single crystals. Kinetic studies on the hydrolysis reaction revealed that the phosphodiesterase activity increases in the order -NO(2)<- Br <- H <- CH(3) when 2,4-bis(dinitrophenyl)phosphate (2,4-bdnpp) was used as substrate, and a linear free energy relationship is found when log(k(cat)/k(0)) is plotted against the Hammett parameter a. However, nuclease activity measurements in the cleavage of double stranded DNA showed that the complexes containing the electron-withdrawing -NO(2) and electron-donating CH3 groups are the most active while the cytotoxic activity of the biomimetics on leukemia and lung tumoral cells is highest for complexes with electron-donating groups.

The Glycosylphosphatidylinositol-Anchored Phosphatase from Spirodela oligorrhiza Is a Purple Acid Phosphatase1

We recently presented clear evidence that the major low-phosphate-inducible phosphatase of the duckweed Spirodela oligorrhiza is a glycosylphosphatidylinositol (GPI)-anchored protein, and, to our knowledge, is the first described from higher plants (N. Morita, H. Nakazato, H. Okuyama, Y. Kim, G.A. Thompson, Jr. [1996] Biochim Biophys Acta 1290: 53–62). In this report the purified 57-kD phosphatase is shown to be a purple metalloenzyme containing Fe and Mn atoms and having an absorption maximum at 556 nm. The phosphatase activity was only slightly inhibited by tartrate, as expected for a purple acid phosphatase (PAP). Furthermore, the protein cross-reacted with an anti-Arabidopsis PAP antibody on immunoblots. The N-terminal amino acid sequence of the phosphatase was very similar to those of Arabidopsis, red kidney bean (Phaseolus vulgaris), and soybean (Glycine max) PAP. Extracts of S. oligorrhiza plants incubated with the GPI-specific precursor [3H]ethanolamine were treated with antibodies raised against the purified S. oligorrhiza phosphatase. Radioactivity from the resulting immunoprecipitates was specifically associated with a 57-kD band on sodium dodecyl sulfate-polyacrylamide gels. These results, together with previous findings, strongly indicate that the GPI-anchored phosphatase of S. oligorrhiza is a PAP.

Direct electrochemistry of porcine purple acid phosphatase (uteroferrin)

Cyclic voltammetry of the non-heme diiron enzyme porcine purple acid phosphatase (uteroferrin, Uf) has been reported for the first time. Totally reversible one-electron oxidation responses (Fe-III-Fe-II --> Fe-III-Fe-III) are seen both in the absence and in the presence of weak competitive inhibitors phosphate and arsenate, and dissociation constants of these oxoanion complexes formed with uteroferrin in its oxidized state (Uf(o)) have been determined. The effect of pH on the redox potentials has been investigated in the range 3 < pH < 6.5, enabling acid dissociation constants for Uf(o) and its phosphate and arsenate complexes to be calculated.

Phosphotyrosyl peptides and analogues as substrates and inhibitors of purple acid phosphatases

Purple acid phosphatases are metal-containing hydrolases. While their precise biological role(s) is unknown, the mammalian enzyme has been linked in a variety of biological circumstances (e.g., osteoporosis) with increased bone resorption. Inhibition of the human enzyme is a possible strategy for the treatment of bone-resorptive diseases such as osteoporosis. Previously, we determined the crystal structure of pig purple acid phosphatase to 1.55 Angstrom and we showed that it is a good model for the human enzyme. Here, a study of the pH dependence of its kinetic parameters showed that the pig enzyme is most efficient at pH values similar to those encountered in the osteoclast resorptive space. Based on the observation that phosphotyrosine-containing peptides are good substrates for pig purple acid phosphatase, peptides containing a range of phosphotyrosine mimetics were synthesized. Kinetic analysis showed that they act as potent inhibitors of mammalian and plant purple acid phosphatases, with the best inhibitors exhibiting low micromolar inhibition constants at pH 3-5. These compounds are thus the most potent organic inhibitors yet reported for the purple acid phosphatases. (C) 2004 Published by Elsevier Inc.

Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K-m) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-Angstrom resolution (final R-free value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to A protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of similar to 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the similar to 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans. (c) 2006 Elsevier B.V. All rights reserved.

Inhibition studies of purple acid phosphatases: Implications for the catalytic mechanism

Purple acid phosphatases (PAPs) belong to the family of binuclear metallohydrolases and catalyse the hydrolysis of a large group of phosphoester substrates at acidic pH. Despite structural conservation in their active sites PAPs appear to display mechanistic versatility. Here, aspects of the catalytic mechanism of two PAPs are investigated using the inhibitors vanadate and fluoride as probes. While the magnitude of their vanadate inhibition constants are similar the two enzymes differ with respect to the mode of inhibition; vanadate interacts in a non-competitive fashion with pig PAP (K-i = 40 mu mol L-1) while it inhibits red kidney bean PAP competitively (K-i = 30 mu mol L-1). Similarly, fluoride also acts as a competitive inhibitor for red kidney bean PAP, independent of pH, while the inhibition of pig PAP by fluoride is uncompetitive at low pH and non-competitive at higher pH, independent of metal ion composition. Furthermore, while fluoride acts as a slow-binding inhibitor in pig PAP it binds rapidly to the catalytic site of the red kidney bean enzyme. Since vanadate and fluoride are proposed to act as transition state and nucleophile mimics, respectively, the observed differences in inhibition kinetics indicate subtle but distinct variations in the reaction mechanism of these enzymes.