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), p yrophosphate, tripolyphosphate, and adenosine 5'-triphosphate (ATP), w ith the Fe(II)Fe(III) form of purple acid phosphatase (PAP(r)) from po rcine uteri (uteroferrin) have been studied by monitoring absorbance c hanges for the iron(III) chromophore at 620 nm. Stopped-flow rate cons tants are independent of total [PO4] (10-50 mM), and decrease with inc reasing 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 bridgin g 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 al pha-naphthyl phosphate, which maximises at pH 4.9. The full mechanism requires participation of Fe(III)-OH, which substitutes into the phosp hate moiety thus bringing about hydrolysis. The concentration of the l atter peaks at pH 4.9, and possible reasons for the decrease in activi ty at pH >4.9 are given. Rate constants at maximum activity are of mag nitude almost-equal-to 0.5 s-1 only, with no very strong discriminatio n between the reagents used. Equilibration steps in which the phosphat e can if necessary be recycled to bring about hydrolysis are proposed. For the pH range studied the final product has a bridging HPO42- liga nd. Trimethyl phosphate with only one oxo group does not appear to rea ct at the Fe(III), but inhibits reaction with H2PO4- possibly by co-or dinating 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(II I) consistent with the decrease in reduction potential from 367 and 18 3 mV. Rate constants are independent of [H2PO4-] and pH.