The divalent metal ion in the active site of uteroferrin modulates substrate binding and catalysis

Mitic, Natasa and Hadler, Keiran S. and Gahan, Lawrence R. and Hengge, Alvan C. and Schenk, Gerhard (2010) The divalent metal ion in the active site of uteroferrin modulates substrate binding and catalysis. Journal of the American Chemical Society, 132 (20). pp. 7049-6054. ISSN 0002-7863

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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 Fe3+-Fe2+ pig PAP (uteroferrin; Uf) and its Fe3+-Mn2+ derivative to investigate the effect of metal ion substitution on the mechanism of catalysis. Replacement of the Fe2+ by Mn2+ 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 Fe3+ 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 Fe3+ 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 Fe3+. The faster rates of interaction with the Fe3+ site in the Fe3+-Mn2+ derivative than the native Fe3+-Fe2+ 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.

Item Type: Article
Additional Information: Preprint version of original published article. The definitive version is available at the Journal of the American Chemical Society, 2010, 132 (20), pp 7049–7054, DOI: 10.1021/ja910583y . Copyright © 2010 American Chemical Society
Keywords: divalent metal ion; uteroferrin; substrate binding; catalysis;
Academic Unit: Faculty of Science and Engineering > Chemistry
Item ID: 3673
Depositing User: Gary Schenk
Date Deposited: 29 May 2012 15:36
Journal or Publication Title: Journal of the American Chemical Society
Publisher: American Chemical Society
Refereed: Yes

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