Hadler, Kieran S. and Tanifum, Eric A. and Hsu-Chen Yip, Sylvia and Mitic, Natasa and Guddat, Luke W. and Jackson, Colin J. and Gahan, Lawrence R. and Nguyen, Kelly and Carr, Paul D. and Ollis, David L. and Hengge, Alvan C. and Larrabee, James A. and Schenk, Gerhard
Substrate-Promoted Formation of a Catalytically Competent
Binuclear Center and Regulation of Reactivity in a
Glycerophosphodiesterase from Enterobacter aerogenes.
Journal of the American Chemical Society, 130 (43).
The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous binuclear
metallohydrolase that catalyzes the hydrolysis of mono-, di-, and triester substrates, including some
organophosphate pesticides and products of the degradation of nerve agents. GpdQ has attracted recent
attention as a promising enzymatic bioremediator. Here, we have investigated the catalytic mechanism of
this versatile enzyme using a range of techniques. An improved crystal structure (1.9 Å resolution) illustrates
the presence of (i) an extended hydrogen bond network in the active site, and (ii) two possible nucleophiles,
i.e., water/hydroxide ligands, coordinated to one or both metal ions. While it is at present not possible to
unambiguously distinguish between these two possibilities, a reaction mechanism is proposed whereby
the terminally bound H2O/OH- acts as the nucleophile, activated via hydrogen bonding by the bridging
water molecule. Furthermore, the presence of substrate promotes the formation of a catalytically competent
binuclear center by significantly enhancing the binding affinity of one of the metal ions in the active site.
Asn80 appears to display coordination flexibility that may modulate enzyme activity. Kinetic data suggest
that the rate-limiting step occurs after hydrolysis, i.e., the release of the phosphate moiety and the
concomitant dissociation of one of the metal ions and/or associated conformational changes. Thus, it is
proposed that GpdQ employs an intricate regulatory mechanism for catalysis, where coordination flexibility
in one of the two metal binding sites is essential for optimal activity.
||The definitive version of this article is published at the Journal of the American Chemical Society, 2008, 130 (43), pp 14129–14138, DOI: 10.1021/ja803346w . Copyright © 2008 American Chemical Society
||Substrate-Promoted; Formation; Catalytically Competent;
Binuclear Center; Regulation; Reactivity; Glycerophosphodiesterase; Enterobacter aerogenes;
||Faculty of Science and Engineering > Chemistry
||29 May 2012 15:29
|Journal or Publication Title:
||Journal of the American Chemical Society
||American Chemical Society
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