Direct Electrochemistry of Porcine Purple Acid Phosphatase (Uteroferrin)

Bernhardt, Paul V.; Schenk, Gerhard; Wilson, Gregory J.

doi:10.1021/bi0490338

Cited by 51 publications

(52 citation statements)

References 41 publications

(70 reference statements)

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“…1) [3][4][5]. Mammalian PAPs are easily and reversibly oxidized to the inactive diferric form owing to the low redox potential (approximately 340 mV) of the divalent iron [6,7], suggesting that the PAP activity may be regulated in vivo by reversible oxidation/reduction of the active site.…”

Section: Introductionmentioning

confidence: 99%

Phosphate ester cleavage promoted by a tetrameric iron(III) complex

et al. 2010

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The purple acid phosphatases (PAPs) are the only binuclear metallohydrolases where the necessity for a heterovalent active site [Fe(III)-M(II) (M is Fe, Zn or Mn)] for catalysis has been established. The paradigm for the construction of PAP biomimetics, both structural and functional, is that the ligands possess characteristics which mimic those of the donor sites of the metalloenzyme and permit discrimination between trivalent and divalent metal ions. The donor atom set of the ligand 2-((2-hydroxy-5-methyl-3-((pyridin-2-ylmethylamino)methyl)benzyl) (2-hydroxybenzyl)amino)acetic acid (H 3 HPBA) mimics that of the active site of PAP although the iron(III) complex of this ligand has been characterized as the tetramer [Fe 4 (HPBA) 2 (l-CH 3 COO) 2 (l-O)(l-OH)(OH 2 ) 2 ]ClO 4 Á5H 2 O. The phosphoesterase-like activity of the complex in 1:1 acetonitrile/water has now been investigated using the substrate 2,4-bis(dinitrophenyl)phosphate. The pH dependence of the catalytic rate revealed a non-symmetric bellshaped profile, with a finite but non-zero rate at high pH. Unlike the traditional approach usually employed to analyse these bell-shaped profiles, the approach used here involved incorporating additional species which contribute to the overall activity. Employing this approach, we show that the complex has a k cat of 1.6 (±0.2) 9 10 -3 s -1 , three kinetically relevant pK a values of 5.3, 6.2 and 8.4, with K M of 7.4 ± 0.6 mM. The kinetic parameters are similar to those reported for heterovalent PAP biomimetics. Additionally, it is observed that, unlike the enzyme, the oxidation state is not the determining factor for catalytic activity.

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Section: Introductionmentioning

confidence: 99%

Phosphate ester cleavage promoted by a tetrameric iron(III) complex

et al. 2010

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“…PAPs catalyze the hydrolysis of a broad range of phosphorylated substrates at acidic to neutral pH, and they require a heterovalent bimetallic active site for reactivity [1,2]. PAPs isolated from mammalian organisms are approximately 35 kDa monomers with a redox-active Fe(III)Fe(II/III) center and a highly conserved amino acid sequence with at least 85% identity across species [1][2][3][4][5]. Homodimeric plant PAPs, extracted from red kidney bean, soybean and sweet potato [6][7][8], have a subunit molecular mass of approximately 55 kDa, and the amino acid sequences are homologous, sharing at least 65% identity [5,7].…”

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confidence: 99%

Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics

et al. 2007

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Purple acid phosphatases (PAPs) are a group of heterovalent binuclear metalloenzymes that catalyze the hydrolysis of phosphomonoesters at acidic to neutral pH. While the metal ions are essential for catalysis, their precise roles are not fully understood. Here, the Fe(III)Ni(II) derivative of pig PAP (uteroferrin) was generated and its properties were compared with those of the native Fe(III)Fe(II) enzyme. The k cat of the Fe(III)Ni(II) derivative (approximately 60 s -1 ) is approximately 20% of that of native uteroferrin, and the Ni(II) uptake is considerably faster than the reconstitution of full enzymatic activity, suggesting a slow conformational change is required to attain optimal reactivity. An analysis of the pH dependence of the catalytic properties of Fe(III)Ni(II) uteroferrin indicates that the l-hydroxide is the likely nucleophile. Thus, the Ni(II) derivative employs a mechanism similar to that proposed for the Ga(III)Zn(II) derivative of uteroferrin, but different from that of the native enzyme, which uses a terminal Fe(III)-bound nucleophile to initiate catalysis. Binuclear Fe(III)Ni(II) biomimetics with coordination environments similar to the coordination environment of uteroferrin were generated to provide both experimental benchmarks (structural and spectroscopic) and further insight into the catalytic mechanism of hydrolysis. The data are consistent with a reaction mechanism employing an Fe(III)-bound terminal hydroxide as a nucleophile, similar to that proposed for native uteroferrin and various related isostructural biomimetics. Thus, only in the uteroferrin-catalyzed reaction are the precise details of the catalytic mechanism sensitive to the metal ion composition, illustrating the significance of the dynamic ligand environment in the protein active site for the optimization of the catalytic efficiency.

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“…The distinctive purple colour of these enzymes is due to a metal to ligand charge transfer from a tyrosine phenolate to a chromophoric Fe(III) (2,3). The cornerstone of the active site of PAP is the presence of two metal ions; Fe(III) is always present in the chromophoric site, while the second site can be occupied by a redox active Fe(II ⁄ III) in mammals (4,5) or a Zn(II) or Mn(II) in plants (6,7). Mammalian PAP is a 35 kDa monomeric protein also known as tartrate-resistant acid phosphatase (TRAP or TRAcP).…”

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Identification of Purple Acid Phosphatase Inhibitors by Fragment‐Based Screening: Promising New Leads for Osteoporosis Therapeutics

et al. 2012

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Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with K i values in the 30-60 lM range. Ligand efficiency values are 0.39-0.44 kcal ⁄ mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3-2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads.Key words: X-ray, crystallography, drug design, fragment screening, purple acid phosphatase, osteoporosis Purple acid phosphatases (PAP) are metalloenzymes that hydrolyse phosphate esters and anhydrides, generally at low pH values (1). The distinctive purple colour of these enzymes is due to a metal to ligand charge transfer from a tyrosine phenolate to a chromophoric Fe(III) (2,3). The cornerstone of the active site of PAP is the presence of two metal ions; Fe(III) is always present in the chromophoric site, while the second site can be occupied by a redox active Fe(II ⁄ III) in mammals (4,5) or a Zn(II) or Mn(II) in plants (6,7). Mammalian PAP is a 35 kDa monomeric protein also known as tartrate-resistant acid phosphatase (TRAP or TRAcP). In contrast, plant PAP is a 110 kDa homodimer, with each subunit consisting of two domains, an N-terminal one whose function is unknown and a catalytic C-terminal domain that strongly resembles the overall structure of the mammalian enzyme (1). Crystal structures of human (8), pig (9), rat (10,11) and plant PAPs (12,13) have been determined and show that the amino acid ligands of the metal ions are completely conserved across plant and animal PAPs, but there are some differences in the identities of the residues that line the active site.A number of biological roles for PAP have been proposed. These include (i) the transport of iron from the mother to the developing foetus during gestation (14); (ii) bone resorption in osteoclasts (evidence for this activity is derived from the effect of PAP on osteoclasts that were cultured on cortical bone slices (15) and from t...

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Direct Electrochemistry of Porcine Purple Acid Phosphatase (Uteroferrin)

Cited by 51 publications

References 41 publications

Phosphate ester cleavage promoted by a tetrameric iron(III) complex

Phosphate ester cleavage promoted by a tetrameric iron(III) complex

Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics

Identification of Purple Acid Phosphatase Inhibitors by Fragment‐Based Screening: Promising New Leads for Osteoporosis Therapeutics

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