The divalent metal ion in the active site of uteroferrin modulates substrate binding and catalysis.
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 mu-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.
Mitić
Natasa
N
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland, 4072, Australia.
Hadler
Kieran S
KS
Gahan
Lawrence R
LR
Hengge
Alvan C
AC
Schenk
Gerhard
G
D000135:Acid Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007501:Iron; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008345:Manganese; D011485:Protein Binding; D013552:Swine; D000071681:Tartrate-Resistant Acid Phosphatase
D007527:Isoenzymes; D008345:Manganese; D007501:Iron; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase