Evidence for nonbridged coordination of p-nitrophenyl phosphate to the dinuclear Fe(III)-M(II) center in bovine spleen purple acid phosphatase duringenzymatic turnover

The pH dependence of the catalytic parameters k(cat) and K-M has been deter mined for the Fe(III)Fe(II)- and Fe(III)Zn(II)-forms of bovine spleen purpl e 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(c at) vs pH profile. Two different EPR spectra were detected for the phosphat e complex of the mixed-valent diiron enzyme; their relative amounts depende d on the pH, with an apparent pK(a) of 6. The EPR spectra of Fe(III)Fe(II)- BSPAP . PO4 and Fe(III)Zn(II)BSPAP . PO4 at pH 5.0 are similar to those pre viously reported for Fe(III)Fe(II)-Uf . PO4 and Fe(III)Zn(II)-Uf . PO4 comp lexes at pH 5.0. At higher pH, a new Fe(III)Fe(II)-BSPAP . PO4 species is f ormed, 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 phos phate up to 30 mM at pH 6.5, suggesting that phosphate binds only to the sp ectroscopically silent Zn(II). To determine whether the phosphate complexes were good structural models for the enzyme substrate complexes, these comp lexes were studied using rapid-freeze EPR and stopped-flow optical spectros copy. 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 simila r, but not identical, to that of the corresponding phosphate complex, both at pH 5 and pH 6.5. We prepose that both phosphate and p-NPP bridge the two metal ions at low pH. At higher pH where the enzyme is optimally active, w e 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 divale nt metal ion.