Oxidation of the reduced (pink) phosphate-free bovine spleen acid phosphatase with 1.5 mol HzOz or sodium peroxodisulfate/mol, in the presence of Mes or Bistris pH 5, leads to a species with an absorption maximum at 558 nm. Addition of acetate or oxidation in the presence of acetate buffer engenders a species with a maximum at 550 nm. Addition of phosphate to both species shifts the maximum immediately to 540 nm; this is the species also found after preparation from the spleen. The assumption that these species represent strongly bidentatebinding hydroxo, acetato and phosphato complexes of the Fe(II1)-Fe(II1) system is supported by replacement reactions with other ligating oxoanions followed by their typical spectral shifts. These oxoanion complexes cannot be dissociated by gel filtration; this is possible only after reduction to the Fe(I1)-Fe(II1) system. The oxidized species without EPR signals below g values of 2 still reveals 5% activity which cannot be reduced to zero even in the presence of higher concentrations of peroxodisulfate. The pH optimum of the reaction with a-naphthyl phosphate shifts from 5.9 to 5.3 in the oxidized species.The apparent pK values around 4.5 as derived from the pH dependence of activity, of the EPR spectra, and the spectral shifts of the phosphate-saturated reduced and oxidized species are assigned to an aquo/hydroxo equilibrium at the Fe(Il1) or an equilibrium, where the phosphato ligand is replaced by a hydroxo ligand. A reaction mechanism is proposed in which a hydroxo ligand at the chromophoric Fe(II1) attacks the phosphoric acid ester group only when that is monoprotonated and pre-oriented by electrostatic interaction with the nonchromophoric metal ion. Binding and inhibition studies with the oxoanions indicate that they compete with the catalytically active hydroxo group of the reduced and oxidized enzyme with nearly the same inhibition constants. Catalysis is not affected by the oxoanions which replace the additional p-hydroxo ligand in the 558-nmabsorbing Fe(II1)-Fe(II1) species. In contrast to hemerythrin and ribonucleotide reductase, a binuclear iron center is proposed for the purple acid phosphatase, which is bridged by a carboxylato and two aquo/hydroxo groups, but without a p-0x0 bridge.Mammalian purple tartrate-resistant acid phosphatases (PAP) [l, 21 have been isolated from porcine uterus [3], bovine spleen [4, 51, rat spleen [6] and rat bone [7]. They are glycoproteins [4-81 with molecular masses of 35 kDa with a monomeric peptide structure. The chain of the bovine enzyme seems to be split into two nonidentical subunits [5]

In order to perform Mossbauer studies, Zn(I1) in the Fe(1II)-Zn(1I) purple acid phosphatase of the red kidney bean has been exchanged by incubating the semiapoenzyme with 57Fe(II). The resulting Fe(III)-s7Fe(II) enzyme has 125% activity, compared with that of the Zn(II) enzyme. It can be oxidized by H,O, or peroxydisulfate to the Fe(III)-57Fe(III) species with a 30-times lower activity. Incubation of the metal-free apoenzyme with s7Fe(II) in the presence of 0, leads to the "Fe(II1)- . They were reported to include essentially manganese. Recent studies suggest, however, that at least the sweet potato phosphatase is an iron enzyme [lo], thus, in regard to the active center, it might correspond to the mammalian enzymes.

Purple acid phosphatase of the common bean Phaseolus vulgaris is a homodimeric 110-kDa glycoprotein with a Fe(II1)-Zn(I1) center in the active site of each monomer. After exchange of Zn(I1) for Fe(II), the enzyme spectroscopically and kinetically resembles the mammalian purple acid phosphatases with Fe(II1)-Fe(11) centers in monomeric 35-kDa proteins. The kidney bean enzyme consists of 432 amino acids/monomer with five N-glycosylated asparagine residues. The complete amino acid sequence was determined by a combination of matrix-assisted laser desorptiod ionization mass spectrometry (MALDI-MS) and classical sequencing methods. Our strategy involved mass determination and sequence analysis of all cyanogen-bromide-generated fragments by automated Edman degradation. Limited cleavages with cyanogen bromide were performed to obtain fragments containing still uncleaved Met-Xaa linkages. MALDI mass spectra of these products allowed the characterization of each fragment and the determination of the order of the cyanogen bromide fragments in the intact protein without producing overlapping peptides. For one large 30-kDa methionine-free fragment, the alignment of the Edman-degraded tryptic peptides was obtained by MALDI-MS analysis and enzymic microscale peptide laddering of overlapping Glu-Cgenerated fragments. The employed strategy shows that the classical method, in combination with modern mass spectrometry, is an attractive approach for primary structure determination in addition to the DNA sequencing method.Purple acid phosphatases (PAP) hydrolysing activated phosphoric acid esters and anhydrides have in common a two-metal center with a tyrosine+Fe(III) charge transfer transition which is responsible for a broad absorption band in the range 500-600 nm and a typical resonance Raman spectrum.

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