Showing papers on "Purple acid phosphatases published in 1999"

Biochemical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties

Abstract: The phosphatases are a diverse class of enzymes. According to one classification, alkaline phosphatases, purple acid phosphatases, high-molecular-weight acid phosphatases, low-molecular-weight acid phosphatases, and protein phosphatases can be distinguished (13). These classes differ in their pH optima, metal ion requirements, substrate specificities, and possibly even reaction mechanisms. The phytases (myo-inositol hexakisphosphate phosphohydrolases; EC 3.1.3.8 and 3.1.3.26) are a subfamily of the high-molecular-weight histidine acid phosphatases. The phytase reaction mechanism is a two-step mechanism which includes a covalent phosphohistidine adduct as an obligatory reaction intermediate (6). Phytases are found naturally in plants and microorganisms, particularly fungi (for a review see reference 15). They catalyze phosphate monoester hydrolysis of phytic acid (myo-inositol hexakisphosphate), which results in the stepwise formation of myo-inositol pentakis-, tetrakis-, tris-, bis-, and monophosphates, as well as the liberation of inorganic phosphate. Phytic acid is the major storage form of phosphorus in plant seeds and, thus, in seed-based animal feed (for reviews see references 1 and 8). Monogastric animals, such as pigs and poultry, are not able to utilize phytic acid phosphorus, since they have only low levels of phytase activity in their digestive tracts and since phytic acid cannot be resorbed. Therefore, pig and poultry feed commonly is supplemented with either inorganic phosphate or a phytase of fungal origin. Despite considerable economic interest, only limited data on the catalytic properties of fungal phytases are available. In order to get an impression of the natural diversity of phytases, the enzymatic properties of six fungal phytases (phytases from Aspergillus niger, two strains of Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila) and of Escherichia coli phytase were characterized in more detail by addressing the following questions. (i) What are the specific activities and pH optima of wild-type phytases? (ii) What are the kinetics of phytic acid degradation, and what are the end products? (iii) Does the substrate specificity profile correlate with the results of in vitro experiments performed to determine phosphate liberation from feed samples? And (iv) what is the potential influence of modulators of enzymatic activity?

A type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and by some other types of phosphate mobilising/oxidative stress conditions.

Abstract: Low phosphorous availability, a common condition of many soils, is known to stimulate phosphatase activity in plants; however, the molecular details of this response remain mostly unknown. We purified and sequenced the N-terminal region of a phosphate starvation induced acid phosphatase (AtACP5) from Arabidopsis thaliana, and cloned its cDNA and the corresponding genomic DNA. The nucleotide sequence of the cDNA predicted that AtACP5 is synthesised as a 338 amino acid-long precursor with a signal peptide. AtACP5 was found to be related to known purple acid phosphatases, especially to mammal type 5 acid phosphatases. Other similarities with purple acid phosphatases, which contain a dinuclear metal centre, include the conservation of all residues involved in metal ligand binding and resistance to tartrate inhibition. In addition, AtACP5, like other type 5 acid phosphatases, displayed peroxidation activity. Northern hybridisation experiments, as well as in situ glucuronidase (GUS) activity assays on transgenic plants harbouring AtACP5:GUS translational fusions, showed that AtACP5 is not only responsive to phosphate starvation but also to ABA and salt stress. It is also expressed in senescent leaves and during oxidative stress induced by H2O2, but not by paraquat or salicylic acid. Given its bifunctionality, as it displays both phosphatase and peroxidation activity, we propose that AtACP5 could be involved in phosphate mobilisation and in the metabolism of reactive oxygen species in stressed or senescent parts of the plant.

Binuclear Metal Centers in Plant Purple Acid Phosphatases: Fe-Mn in Sweet Potato

Abstract: Purple acid phosphatases comprise a family of binuclear metal-containing acid hydrolases, representatives of which have been found in animals, plants, and fungi. The goal of this study was to characterize purple acid phosphatases from sweet potato tubers and soybean seeds and to establish their relationship with the only well-characterized plant purple acid phosphatase, the FeIII-ZnII-containing red kidney bean enzyme. Metal analysis indicated the presence in the purified sweet potato enzyme of 1.0 g-atom of iron, 0.6-0.7 g-atom of manganese, and small amounts of zinc and copper. The soybean enzyme contained 0.8-0.9 g-atom of iron, 0.7-0.8 g-atom of zinc per subunit, and small amounts of manganese, copper, and magnesium. Both enzymes exhibited visible absorption maxima at 550-560 nm, with molar absorption coefficients of 3200 and 3300 M(-1) cm(-1), respectively, very similar to the red kidney bean enzyme. Substrate specificities were markedly different from those of the red kidney bean enzyme. A cloning strategy was developed based on N-terminal sequences of the sweet potato and soybean enzymes and short sequences around the conserved metal ligands of the mammalian and red kidney bean enzymes. Three sequences were obtained, one from soybean and two from sweet potato. All three showed extensive sequence identity (>66%) with red kidney bean purple acid phosphatase, and all of the metal ligands were conserved. The combined results establish that these enzymes are binuclear metalloenzymes: Fe-Mn in the sweet potato enzyme and Fe-Zn in soybean. The sweet potato enzyme is the first well-defined example of an Fe-Mn binuclear center in a protein.

Tartrate-resistant purple acid phosphatase is synthesized as a latent proenzyme and activated by cysteine proteinases.

Abstract: Purple acid phosphatases (PAPs) are binuclear acid metallohydrolases also referred to as tartrate-resistant acid phosphatases (TRAPs) or type 5 acid phosphatases. The cDNA sequences of TRAP/PAP enzymes from different species and organs indicate that these enzymes are translated as monomeric polypeptides of approx. 35 kDa, contrasting with the predominantly two-subunit structure observed in purified enzyme preparations. In the present study we have compared certain structural and enzyme-kinetic properties of recombinant rat PAP (monomeric) with those of the native rat bone TRAP/PAP enzyme (two-subunit), and examined effects on these parameters by cleaving the monomeric recombinant PAP with the serine proteinase trypsin or the cysteine proteinases papain or cathepsin B. Cleavage with trypsin resulted in a moderate activation of the recombinant enzyme and shifted the pH optimum to a slightly more basic value (5.0-5.5). Cleavage with papain resulted in complete activation and conferred similar properties to those of the bone PAP variant with regard to pH optimum (5.5-6.0) and sensitivity to reducing agents, as well as in the sizes of the subunits. Substrate specificity studies showed that the two-subunit bone PAP was considerably more active than the monomeric recombinant rat PAP towards a variety of serine-, threonine- and tyrosine-phosphorylated substrates. Of these substrates, bovine milk osteopontin seemed to be the most readily dephosphorylated substrate. In conclusion, the results suggest that the monomeric form of PAP represent a latent proenzyme with low enzymic activity towards both tyrosine- and serine/threonine-containing phosphorylated substrates. Besides being implicated in the catabolism of the extracellular matrix, members of the cysteine proteinase family might also exert a regulatory role in degradative processes involving the PAP enzymes by converting the newly synthesized PAPs to enzymically active and microenvironmentally regulated species.

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

Abstract: 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.

Fluoride inhibition of bovine spleen purple acid phosphatase: characterization of a ternary enzyme-phosphate-fluoride complex as a model for the active enzyme-substrate-hydroxide complex.

Abstract: Purple acid phosphatases (PAPs) employ a dinuclear Fe3+Fe2+ or Fe3+Zn2+ center to catalyze the hydrolysis of phosphate monoesters. The interaction of fluoride with bovine spleen purple acid phosphatase (BSPAP) has been studied using a combination of steady-state kinetics and spectroscopic methods. For FeZn−BSPAP, the nature of the inhibition changes from noncompetitive at pH 6.5 (Ki(comp) ≈ Ki(uncomp) ≈ 2 mM) to uncompetitive at pH 5.0 (Ki(uncomp) = 0.2 mM). The inhibition constant for AlZn−BSPAP at pH 5.0 (Ki = 3 μM) is ∼50−70-fold lower than that observed for both FeZn−BSAP and GaZn−BSPAP, suggesting that fluoride binds to the trivalent metal. Fluoride binding to the enzyme−substrate complex was found to be remarkably slow; hence, the kinetics of fluoride binding were studied in some detail for FeZn−, AlZn−, and FeFe−BSPAP at pH 5.0 and for FeZn−BSPAP at pH 6.5. Since the enzyme kinetics studies indicated the formation of a ternary enzyme−substrate-fluoride complex, the binding of fluoride to FeZn−BSPAP...

Probing the Role of the Trivalent Metal in Phosphate Ester Hydrolysis: Preparation and Characterization of Purple Acid Phosphatases Containing AlIIIZnII and InIIIZnII Active Sites, Including the First Example of an Active Aluminum Enzyme

Abstract: Purple acid phosphatases contain a dinuclear Fe3+M2+ center in their active site (M = Fe2+ or Zn2+). To resolve the specific role of the ferric ion in catalysis, a series of metal-substituted forms of bovine spleen purple acid phosphatase (BSPAP) of general formula MIIIZnII-BSPAP has been prepared, in which the trivalent metal ion was systematically varied (MIII = Al, Fe, Ga, and In). The activity of the AlZn-BSPAP form was only slightly lower (kcat ≈ 2000 s-1) than that of the previously reported GaZn and FeZn forms (kcat ≈ 3000 s-1). The InZn form was inactive. The kinetics parameters and pH profile of AlZn-BSPAP were remarkably similar to those of FeZn-BSPAP and GaZn-BSPAP, but AlZn-BSPAP was readily distinguished from the GaZn and FeZn forms by its 50−70-fold lower inhibition constant for fluoride. These results are not, at first sight, consistent with intrinsic properties of the trivalent metal ions as they are known from coordination chemistry. In particular, aluminum has generally been believed to ...

Evidence for Nonbridged Coordination of p-Nitrophenyl Phosphate to the Dinuclear Fe(III)−M(II) Center in Bovine Spleen Purple Acid Phosphatase during Enzymatic Turnover

Abstract: The pH dependence of the catalytic parameters kcat and KM has been determined for the Fe(III)Fe(II)- and Fe(III)Zn(II)-forms of bovine spleen purple acid phosphatase (BSPAP). The parameter kcat was...

Structure, Properties and Reactivity of the FeIIFeIII and ZnIIFeIII Purple Acid Phosphatases

Abstract: This microreview describes the structure, properties and mechanisms of the purple acid phosphatases (PAP). The enzyme is isolated from mammalian, plant and bacterial sources. X-ray structural information is now available for the enzyme from pig (uteroferrin), rat and kidney beans. Features of the mechanism are the concerted action of a labile MII centre (FeII or ZnII) alongside a more inert FeIII. The latter is effective as a conjugate-base FeOH2+, which initiates hydrolysis at the MII-bound phosphate ester by a process involving OH− replacement of OR− at the PV. Histidine residues near to the active site help bind the phosphate and are involved in the release of OR−. Effects of replacement of the FeII by MnII, CoII, NiII, CuII and ZnII, and of FeIII by GaIII,AlIII and InIII have been studied. The mechanistic role of the ZnIIZnII combination in alkaline phosphatases, and other related dinuclear centres is also considered.

Crystallization and preliminary X-ray diffraction data for a purple acid phosphatase from sweet potato

Abstract: Purple acid phosphatase from sweet potato is a homodimer of 110 kDa. Two forms of the enzyme have been characterized. One contains an Fe–Zn centre similar to that previously reported for red kidney bean purple acid phosphatase. Another isoform, the subject of this work, is the first confirmed example of an Fe–Mn-containing enzyme. Crystals of this protein have been grown from PEG 6000. They have unit-cell parameters a = b = 118.4, c = 287.4 A and have the symmetry of space group P6522, with one dimer per asymmetric unit. Diffraction data collected using a conventional X-­ray source from a cryocooled crystal extend to 2.90 A resolution. The three-dimensional structure of the enzyme will provide insight into the coordination of this novel binuclear metal centre.

Carboxylate/diphenylphosphate exchanges in asymmetric diiron complexes modeling the purple acid phosphatases enzymes : associated redox processes

Abstract: Redox effects dominate the carboxylate/diphenylphosphate ligand exchange in diiron complexes which model the active site of purple acid phosphatases

Structural and Functional Studies on Model Compounds of Purple Acid Phosphatases and Catechol Oxidases

Abstract: The synthesis, single crystal X-ray crystallographic, magnetic and electrochemical characterization of eight representative symmetric and unsymmetric complexes as structural model compounds for active sites in PAPs is reported. A mixed valent diiron as well as an iron(III)–zinc(II) complex as models for the active, reduced form of mammalian and plant PAPs, respectively, were synthesized and characterized. Five diiron(III) compounds as structural models for the oxidized uteroferrin-phosphato and -arsenato complex and a model for the oxidized form of PAP from beef spleen are reported. In addition to the structural relevance the catalase and peroxidase activity of one of these model complexes is introduced. Further we summarize our recent research concerning synergistic investigations on catechol oxidase and on synthetic copper coordination complexes. The catechol oxidase is an important type 3 copper protein for the activation of dioxygen. The development of low-molecular weight catalysts should facilitate the oxidation of organic substances by O2. In particular the reported copper(II) complexes may serve as structural and functional bioinorganic model compounds for the active sites of dioxygen binding and dioxygen activating copper proteins, respectively. These investigations provided a new X-ray crystallographically characterized type of peroxo copper(II) complexes with a μ4-(η1)4 coordination mode.

Crystallization and preliminary X-ray diffraction studies of mammalian purple acid phosphatase

Abstract: The oxidized form of purple acid phosphatase from pig allantoic fluid has been crystallized in the presence of phosphate using the hanging-drop technique. The crystals belong to the space group P212121 and have unit-cell parameters a = 66.8, b = 70.3, c = 78.7 A. Diffraction data collected from a cryocooled crystal using a conventional X-ray source extend to 1.55 A resolution. A knowledge of the three-dimensional structure of mammalian purple acid phosphatase will aid in understanding the substrate specificity of the enzyme and will be important in the rational design of inhibitors, with potential in the treatment of bone diseases.

Phosphatases, Lewis acids, and vanadium

Abstract: Advances in Zinc Enzyme Models by Small, Mononuclear Zinc (II) Complexes.- Zinc catalysis in metalloproteases.- Modeling the biological chemistry of vanadium: Structural and reactivity studies elucidating biological function.- Vanadium bromoperoxidase and functional mimics.- Metal ions in the mechanism of enzyme-catalysed phosphate monoester hydrolyses.- The Dimetal Center in purple acid phosphatases.

EXAFS studies on the active site of purple acid phosphatase from sweet potato Ipomoea batatas.

Abstract: XAS investigations on the active site of the purple acid phosphatase isolated from the sweet potato Ipomoea batatas provided first information about the structure. Data analysis reveal a dinuclear active site containing an iron and a zinc atom. The first coordination sphere of each metal in the active site consists of six nitrogen/oxygen donor atoms, resulting in a distorted octahedral coordination sphere very similar to the active site of the enzyme isolated from the red kidney bean Phaseolus vulgaris. The XAS spectra were recorded in fluorescence mode at 18 K using a Canberra 13-element germanium solid state detector.

Crystallization and preliminary X-ray diffractiondata for a purple acid phosphatase from sweetpotato

Abstract: Purple acid phosphatase from sweet potato is a homodimer of 110 kDa. Two forms of the enzyme have been characterized. One contains an Fe±Zn centre similar to that previously reported for red kidney bean purple acid phosphatase. Another isoform, the subject of this work, is the ®rst con®rmed example of an Fe±Mn-containing enzyme. Crystals of this protein have been grown from PEG 6000. They have unit-cell parameters a = b = 118.4, c = 287.4 A E and have the symmetry of space group P6522, with one dimer per asymmetric unit. Diffraction data collected using a conventional X-ray source from a cryocooled crystal extend to 2.90 A E resolution. The three-dimensional structure of the enzyme will provide insight into the coordination of this novel binuclear metal centre.

Crystalline form of activated tartrate-resistant and purple acid phosphatase

Abstract: A crystalline form of mammalian TRAP (tartrate-resistant and purple acid phosphatase) is described. The enzyme is activated by cleavage prior to crystallization with a protease and the crystalline form of the mammalian TRAP is capable of being used for X-ray studies.

Mechanistic studies on the diiron center in bovine spleen purple acid phosphatase

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