Purification and characterization of human bone tartrate-resistant acid phosphatase

Allen, Susan H.; Nuttleman, Peter R.; Ketcham, Catherine M; Roberts, R. Michael

doi:10.1002/jbmr.5650040108

Cited by 59 publications

(7 citation statements)

References 33 publications

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“…[1][2][3][22][23][24][25][26] The identity of the divalent metal ion varies with the source of the enzyme. Mammalian PAPs contain an antiferromagnetically coupled binuclear iron center Fe(III)-Fe(II) in the active site, [27][28][29][30][31][32][33] while plant PAPs most typically have Fe(III)-Zn(II) centers, 26,[34][35][36] but an interesting example of an enzymatically active binuclear Fe(III)-Mn(II) center was found in the case of PAP from the sweet potato. 24,25,37 In spite of the scarce similarity in their primary sequences, the active site structure and the residues coordinating the metal ions in the active site are identical in mammalian and plant PAPs, displaying similar enzymatic and spectroscopic properties.…”

Section: Introductionmentioning

confidence: 99%

Atomistic details of the Catalytic Mechanism of Fe(III)−Zn(II) Purple Acid Phosphatase

Alberto

Marino

Ramos

et al. 2010

J. Chem. Theory Comput.

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In the present work, we performed a theoretical investigation of the reaction mechanism of the Fe(III)-Zn(II) purple acid phosphatase from red kidney beans (rkbPAP), using the hybrid density functional theory and employing different exchange-correlation potentials. Characterization of the transition states and intermediates involved and the potential energy profiles for the reaction in different environments (gas phase, protein environment, and water) are reported. Our results show that the Fe(III)-Zn(II)PAP catalyzes the hydrolysis of methylphosphate via direct attack by a bridging metals-coordinated hydroxide leading to the cleavage of the ester bond. From our study emerges that the rate-limiting step of the reaction is the nucleophilic attack followed by the less energetically demanding release of the leaving group. Furthermore, we provide insights into some important points of contention concerning the precatalytic complex and the substrate coordination mode into the active site prior to hydrolysis. In particular: (i) Two models of enzyme-substrate with different orientations of the substrate into the active site were tested to evaluate the possible roles played by the conserved histidine residues (His 202 and His 296); (ii) Different protonation states of the substrate were taken into account in order to reproduce different pH values and to verify its influence on the catalytic efficiency and on the substrate binding mode; (iii) The metals role in each step of the catalytic mechanism was elucidated. We were also able to ascertain that the activation of the leaving group by the protonated His 296 is decisive to reach an optimal catalytic efficiency, while the bond scission without activation requires higher energy to occur.

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Section: Introductionmentioning

confidence: 99%

Atomistic details of the Catalytic Mechanism of Fe(III)−Zn(II) Purple Acid Phosphatase

Alberto

Marino

Ramos

et al. 2010

J. Chem. Theory Comput.

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“…Increase of PAP level in breast cancer cells lead to its physiological function with DNA [22]. Such DNA cleavage activity is shown by RAPase of Fe-1 while Fe-2 RAPase indicates absence of such activity.…”

Section: Dna Cleavage Activitymentioning

confidence: 95%

Dimeric Fe (II, III) complex of quinoneoxime as functional model of PAP enzyme: Mössbauer, magneto-structural and DNA cleavage studies

Salunke‐Gawali

Ahmed

Varret³

et al. 2008

Hyperfine Interact

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Purple acid phosphatase, (PAP), is known to contain dinuclear Fe 2 +2,+3 site with characteristic Fe +3 ← Tyr ligand to metal charge transfer in coordination. Phthiocoloxime (3-methyl-2-hydroxy-1,4-naphthoquinone-1-oxime) ligand L, mimics (His/Tyr) ligation with controlled and unique charge transfers resulting in valence tautomeric coordination with mixed valent diiron site in model compound Fe-1:a molecularly associated dimer of phthiocoloxime synthesized for comparison of charge transfer. 57 Fe Mössbauer studies was used to quantitize unusual valences due to ligand in dimeric Fe-1 and Fe-2 complexes which are supported by EPR and SQUID studies. 57 Fe Mössbauer spectra for Fe-1 at 300 K indicates the presence of two quadrupole split asymmetric doublets due to the differences in local coordination geometries of [Fe +3 ] A and [Fe +2 ] B sites. The hyperfine interaction parameters are δ A = 0.152, ( E Q ) A = 0.598 mm/s with overlapping doublet at δ B = 0.410 and ( E Q ) B = 0.468 mm/s. Due to molecular association tendency of ligand, dimer Fe-2 possesses 100% Fe +3 (h.s.) hexacoordinated configuration with isomer shift δ = 0.408 mm/s. Slightly distorted octahedral symmetry created by NQ CH3ox ligand surrounding Fe +3 (h.s.) state generates small field gradient indicated by quadrupole split E Q = 0.213 mm/s. Decrease of isomer shifts together with variation of quadrupole splits with temperature in Fe-1 dimer compared to Fe-2 is S. Salunke-Gawali et al. result of charge transfers in [Fe 2+2,+3 SQ] complexes. EPR spectrum of Fe-1 shows two strong signals at g 1 = 4.17 and g 2 = 2.01 indicative of S = 3/2 spin state with an intermediate spin of Fe +3 (h.s.) configuration. SQUID data of χ corr m .T were best fitted by using HDVV spin pair model S = 2, 3/2 resulting in antiferromagnetic exchange (J = −13.5 cm −1 with an agreement factor of R = 1.89 × 10 −5 ). The lower J value of antiferromagnetic exchange leads to Fe +3 μ-(OH) Fe +2 bridging in Fe-1 dimer instead of μ-oxo bridge. The intermolecular association through H-bonds may lead to weakly coupled antiferromagnetic interaction between two Fe-2 molecules having Fe +3 (h.s.) centers. Using S = 5/2, 5/2 spin pair model we obtained bestfitted parameters such as J = −12.4 cm −1 , g = 2.3 with R = 3.58 × 10 −5 . Synthetic strategy results in non-equivalent iron sites in Fe-1 dimer analogues to PAP enzyme hence its reconstitution results in pUC-19 DNA cleavage activity, as physiological functionality of APase. It is compared with nuclease activity of Fe-2 RAPase.

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“…1, lane 6), as described also in Ref. 30. When we loaded the preparation onto a ConA-Sepharose column, the 33 kd band went through the column, whereas all of the TRAP activity was bound to it.…”

Section: Purification Of Human Bone Trapmentioning

confidence: 97%

Tartrate-resistant acid phosphatase from human bone: Purification and development of an immunoassay

Halleen

Hentunen

Hellman

et al. 1996

Journal of Bone and Mineral Research

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Tartrate-resistant acid phosphatase (TRAP) was purified 20,000-fold to apparent homogeneity from human bone. The purified enzyme consisted of one 32 kd subunit, which was cleaved by beta-mercaptoethanol into two subunits of 15 kd and 20 kd, as shown by sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The purified enzyme was identified by N-terminal amino acid sequencing, and it was shown to be homologous with previously purified TRAPs from other sources. We developed a polyclonal antiserum against the purified enzyme in mice. In immunohistochemistry, the antiserum recognized osteoclasts from human bone and alveolar macrophages from human lung tissue, but no cells from human spleen tissue. It also stained osteoclasts from rat bone cells cultured on bovine bone slices. Purified TRAP could be inhibited by vanadate and molybdate, but not by tartrate, and it was activated 2-fold by beta-mercaptoethanol. The glycoprotein structure of human bone TRAP was analyzed, and it was shown to contain only high-mannose type carbohydrates. We used the polyclonal antibody to develop a competitive fluorescence immunoassay for measuring serum TRAP concentrations. According to the assay, children have higher serum TRAP concentrations than adults, and postmenopausal women have higher concentrations than premenopausal women. Postmenopausal women also have higher serum TRAP concentrations than postmenopausal women on estrogen replacement therapy.

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Purification and characterization of human bone tartrate-resistant acid phosphatase

Cited by 59 publications

References 33 publications

Atomistic details of the Catalytic Mechanism of Fe(III)−Zn(II) Purple Acid Phosphatase

Atomistic details of the Catalytic Mechanism of Fe(III)−Zn(II) Purple Acid Phosphatase

Dimeric Fe (II, III) complex of quinoneoxime as functional model of PAP enzyme: Mössbauer, magneto-structural and DNA cleavage studies

Tartrate-resistant acid phosphatase from human bone: Purification and development of an immunoassay

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