Abstract
To date, most spectroscopic studies on mammalian purple acid phosphatases (PAPs) have been performed at a single pH, typically pH 5. The catalytic activity of these enzymes is, however, pH dependent, with optimal pH values of 5.5–6.2 (depending on the form). For example, the pH optimum of PAPs isolated as single polypeptides is around pH 5.5, which is substantially lower that of proteolytically cleaved PAPs (ca. pH 6.2). In addition, the catalytic activity of single polypeptide PAPs at their optimal pH values is four to fivefold lower than that of the proteolytically cleaved enzymes. In order to elucidate the chemical basis for the pH dependence of these enzymes, the spectroscopic properties of both the single polypeptide and proteolytically cleaved forms of recombinant human PAP (recHPAP) and their complexes with inhibitory anions have been examined over the pH range 4 to 8. The EPR spectra of both forms of recHPAP are pH dependent and show the presence of three species: an inactive low pH form (pH<pK a,1), an active form (pK a,1<pH<pK a,2), and an inactive high pH form (pH>pK a,2). The pK a,1 values observed by EPR for the single polypeptide and proteolytically cleaved forms are similar to those previously observed in kinetics studies. The spectroscopic properties of the enzyme–phosphate complex (which should mimic the enzyme–substrate complex), the enzyme–fluoride complex, and the enzyme–fluoride–phosphate complex (which should mimic the ternary enzyme–substrate–hydroxide complex) were also examined. EPR spectra show that phosphate binds to the diiron center of the proteolytically cleaved form of the enzyme, but not to that of the single polypeptide form. EPR spectra also show that fluoride binds only to the low pH form of the enzymes, in which it presumably replaces a coordinated water molecule. The binding of fluoride and phosphate to form a ternary complex appears to be cooperative.
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Abbreviations
- PAP:
-
Purple acid phosphatase
- recHPAP:
-
Recombinant purple acid phosphatase from human placenta
- recRPAP:
-
Recombinant purple acid phosphatase from rat bone
- Uf:
-
Purple acid phosphatase from pig uterine fluids
- BSPAP:
-
Purple acid phosphatase from bovine spleen
- PP1:
-
Protein phosphatase 1
- PP2B:
-
Calcineurin
- p-NPP:
-
para-nitrophenylphosphate
- MES:
-
2-[N-morpholino]ethanesulfonic acid
- HEPES:
-
(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid])
- BSA:
-
Bovine serum albumine
- EPR:
-
Electron paramagnetic resonance
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Acknowledgements
This research was supported by a grant from the EU Biotechnology Program (contract B104-CT-98-0385).
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Funhoff, E.G., de Jongh, T.E. & Averill, B.A. Direct observation of multiple protonation states in recombinant human purple acid phosphatase. J Biol Inorg Chem 10, 550–563 (2005). https://doi.org/10.1007/s00775-005-0001-9
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DOI: https://doi.org/10.1007/s00775-005-0001-9