Reactivity of MII metal-substituted derivatives of pig purple acid phosphatase (uteroferrin) with phosphate

The Fe^II of the binuclear Fe^IIFe^III active site of pig purple acid phosphatase (uteroferrin) has been replaced in turn by five M^II ions (Mn^II, Co^II, Ni^II, Cu^II, and Zn^II). An uptake of 1 equiv of M^II is observed in all cases except that of Cu^II, when a second more loosely bound Cu^II is removed by treatment with edta. The products have been characterized by different analytical procedures and by UV-vis spectrophotometry. At 25 °C,/= 0.100 M (NaCI), the nonenzymatic reactions with H₂PO₄ ^- give the μ-phosphato product, and formation constants K/M^-1 show an 8-fold spread at pH 4.9 of 740 (Mn), 165 (Fe), 190 (Co), 90 (Ni), 800 (Cu), 380 (Zn), The variations in K correlate well with stability constants for the complexing of H₂PO₄ ^- and (CH₃O)HPO₃ ^- with M^II hexaaqua ions. At pH 4.9 with [H₂PO₄ ^-] ≥ 3.5 mM rate constants k_obs decrease, and an inhibition process in which a second [H₂PO₄ ^-] coordinates to the dinuclear center is proposed. The mechanism considered accounts for most but not all of the features displayed. Thus K₁ values for the coordination of phosphate to M^II are in the rangelO-60 M^-1, whereas K₂ values for the bridging of the phosphate to Fe^III are in the narrower range 7.8-12.4. From the fits described K_i ∼ 10³ M^-1 for the inhibition step, which is independent of the identity of M^II. Values of k_obs decrease with increasing pH, giving pKa values which are close to 3.8 and independent of M^II (Fe^II, Zn^II, Mn^II). The acid dissociation process is assigned to Fe^III-OH₂ to Fe^III-OH^-, where OH^- is less readily displaced by phosphate.