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.

Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics

“…In agreement with previous studies [40,[45][46][47] pK es2 is assigned to a conserved histidine residue in the second coordination sphere, which is likely to act as a proton donor to the leaving alcohol group during catalysis [14,[47][48][49]. Site-directed mutagenesis studies identified His92 as the likely residue [47].…”

Phosphotyrosyl peptides and analogues as substrates and inhibitors of purple acid phosphatases

“…The presence of a bridging l-hydroxo group in the active purple acid phosphatases from pig, bovine, and red kidney bean is supported by saturation magnetization measurements, which indicated weak exchange coupling [33,34]. A role as the attacking nucleophile has been attributed to this bridging group as an alternative to the hydroxo group coordinated to ferric ion [35][36][37][38]. The pK a of $2 could alternatively be due to the first ionization of the substrate, although we would expect the value to be lower-the first acid dissociation of phenyl phosphate is reported to be 0.3 [25].…”

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

“…For both enzymes the K m values for the substrate p-NPP increase as the pH is increased, an observation which is likely to be due to the deprotonation of a histidine residue in the substrate binding pocket of the enzyme. 16,39 The inhibitory effect of vanadate on the activity of red kidney bean PAP was determined at optimum pH (pH 6.2). At that pH vanadate monomers tend to oligomerise in solution provided the total concentration of the tetraoxo anion is ≥ 0.25 mmol L -1 .…”

“…For both enzymes the K m values for the substrate p-NPP increase as the pH is increased, an observation which is likely to be due to the deprotonation of a histidine residue in the substrate binding pocket of the enzyme. 16,39 Vol. 17, No.…”