The Divalent Metal Ion in the Active Site of Uteroferrin Modulates Substrate Binding and Catalysis

摘要

The purple acid phosphatases (PAP) are binuclear metallohydrolases that catalyze the hydrolysis of a broad range of phosphomonoester substrates. The mode of substrate binding during catalysis and the identity of the nucleophile is subject to debate. Here, we used native Fe~(3+)-Fe~(2+) pig PAP (uteroferrin; Uf) and its Fe~(3+)-Mn~(2+) derivative to investigate the effect of metal ion substitution on the mechanism of catalysis. Replacement of the Fe~(2+) by Mn~(2+) lowers the reactivity of Uf. However, using stopped-flow measurements it could be shown that this replacement facilitates approximately a ten-fold faster reaction between both substrate and inorganic phosphate with the chromophoric Fe~(3+) site. These data also indicate that in both metal forms of Uf, phenyl phosphate hydrolysis occurs faster than formation of a μ-1 ,3 phosphate complex. The slower rate of interaction between substrate and the Fe~(3+) site relative to catalysis suggests that the substrate is hydrolyzed while coordinated only to the divalent metal ion. The likely nucleophile is a water molecule in the second coordination sphere, activated by a hydroxide terminally coordinated to Fe~(3+). The faster rates of interaction with the Fe~(3+) site in the Fe~(3+)-Mn~(2+) derivative than the native Fe~(3+)-Fe~(2+) form are likely mediated via a hydrogen bond network connecting the first and second coordination spheres, and illustrate how the selection of metal ions may be important in fine-tuning the function of this enzyme.