MECHANISM OF THE REACTION OF DIFFERENT PHOSPHATES WITH THE IRON(II)IRON(III) FORM OF PURPLE ACID-PHOSPHATASE FROM PORCINE UTERI (UTEROFERRIN)

Reactions of different phosphates (represented here as PO4), including H2PO4-(as prototype), phenylphosphate (and the p-nitro derivative), pyrophosphate, tripolyphosphate, and adenosine 5'-triphosphate (ATP), with the Fe(II)Fe(III) form of purple acid phosphatase (PAP(r)) from porcine uteri (uteroferrin) have been studied by monitoring absorbance changes for the iron(III) chromophore at 620 nm. Stopped-flow rate constants are independent of total [PO4] (10-50 mM), and decrease with increasing pH (2.5-6.5). At the lower pH a mechanism of rapid PO4 binding to the Fe(II), followed by rate-controlling [PO4]-independent bridging to the Fe(III) with displacement of a co-ordinated H2O, is proposed. Further information comes from experiments on the hydrolysis activity of PAP(r) monitored by the release of alpha-naphthol (323 nm) from alpha-naphthyl phosphate, which maximises at pH 4.9. The full mechanism requires participation of Fe(III)-OH, which substitutes into the phosphate moiety thus bringing about hydrolysis. The concentration of the latter peaks at pH 4.9, and possible reasons for the decrease in activity at pH >4.9 are given. Rate constants at maximum activity are of magnitude almost-equal-to 0.5 s-1 only, with no very strong discrimination between the reagents used. Equilibration steps in which the phosphate can if necessary be recycled to bring about hydrolysis are proposed. For the pH range studied the final product has a bridging HPO42- ligand. Trimethyl phosphate with only one oxo group does not appear to react at the Fe(III), but inhibits reaction with H2PO4- possibly by co-ordinating to the Fe(II). Reaction with the sterically bulky cation [Co(NH3)5(HPO4)]+ is much slower, k = 1.6 x 10(-4) s-1. The HPO4--bridged Fe(II)Fe(III) form is more responsive to air oxidation to Fe(III)Fe(III) consistent with the decrease in reduction potential from 367 and 183 mV. Rate constants are independent of [H2PO4-] and pH.