title: 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 k(cat) and K(M) has been determined for the Fe(III)Fe(II)- and Fe(III)Zn(II)-forms of bovine spleen purple acid phosphatase (BSPAP). The parameter k(cat) was found to be maximal at pH 6.3, and a pK(a) of 5.4-5.5 was obtained for the acidic limb of the k(cat) vs pH profile. Two different EPR spectra were detected for the phosphate complex of the mixed-valent diiron enzyme; their relative amounts depended on the pH, with an apparent pK(a) of 6. The EPR spectra of Fe(III)Fe(II)-BSPAP.PO(4) and Fe(III)Zn(II)-BSPAP.PO(4) at pH 5.0 are similar to those previously reported for Fe(III)Fe(II)-Uf.PO(4) and Fe(III)Zn(II)-Uf.PO(4) complexes at pH 5.0. At higher pH, a new Fe(III)Fe(II)-BSPAP.PO(4) species is formed, with apparent g-values of 1.94, 1.71, and 1.50. The EPR spectrum of Fe(III)Zn(II)-BSPAP does not show significant changes upon addition of phosphate up to 30 mM at pH 6.5, suggesting that phosphate binds only to the spectroscopically silent Zn(II). To determine whether the phosphate complexes were good structural models for the enzyme substrate complexes, these complexes were studied using rapid-freeze EPR and stopped-flow optical spectroscopy. The stopped-flow studies showed the absence of burst kinetics at pH 7.0, which indicates that substrate hydrolysis is rate limiting, rather than phosphate release. The EPR spectrum of Fe(III)Fe(II)-BSPAP.p-NPP is similar, but not identical, to that of the corresponding phosphate complex, both at pH 5 and pH 6.5. We propose that both phosphate and p-NPP bridge the two metal ions at low pH. At higher pH where the enzyme is optimally active, we propose that hydroxide competes with phosphate and p-NPP for coordination to Fe(III) and that both phosphate and p-NPP coordinate only to the divalent metal ion.
authors.affiliation: E.C. Slater Institute, Biocentrum Amsterdam, University of Amsterdam, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Merkx
authors.forename: M W
authors.initials: MW
authors.lastname: Pinkse
authors.forename: B A
authors.initials: BA
authors.lastname: Averill
mesh_terms: D000135:Acid Phosphatase / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry*; D005296:Ferrous Compounds / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D009596:Nitrophenols / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D013053:Spectrophotometry; D013154:Spleen; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry
chemical_list: D005290:Ferric Compounds; D005296:Ferrous Compounds; D006023:Glycoproteins; D046911:Macromolecular Substances; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D015032:Zinc