Hadler, Kieran S. and Mitic, Natasa and Ely, Fernanda and Hanson, Graeme R. and Gahan, Lawrence R. and Larrabee, James A. and Ollis, David L. and Schenk, Gerhard
Structural Flexibility Enhances the Reactivity of the
Bioremediator Glycerophosphodiesterase by Fine-Tuning Its
Mechanism of Hydrolysis.
Journal of the American Chemical Society, 131 (33).
The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) belongs to the family of
binuclear metallohydrolases and has attracted recent attention due to its potential in bioremediation.
Formation of a catalytically competent binuclear center is promoted by the substrate (Hadler et al. J. Am.
Chem. Soc. 2008, 130, 14129). Using the paramagnetic properties of Mn(II), we estimated the Kd values
for the metal ions in the R and sites to be 29 and 344 μM, respectively, in the absence of a substrate
analogue. In its presence, the affinity of the site increases substantially (Kd ) 56 μM), while that of the
R site is not greatly affected (Kd ) 17 μM). Stopped-flow fluorescence measurements identified three distinct
phases in the catalytic turnover, associated with the initial binding of substrate to the active site (kobs1), the
assembly of a catalytically active binuclear center (kobs2), and subsequent slower structural rearrangements
to optimize catalysis (kobs3). These three phases depend on the concentration of substrate ([S]), with kobs1
and kobs2 reaching maximum values at high [S] (354 and 38 s-1, respectively), whereas kobs3 is reduced as
[S] is increased. The kcat for the hydrolysis of the substrate bis(para-nitrophenyl) phosphate (∼1 s-1) gradually
increases from the moment of initiating the reaction, reaching a maximum when the structural change
associated with kobs3 is complete. This structural change is mediated via an extensive hydrogen-bond network
that connects the coordination sphere with the substrate binding pocket, as demonstrated by mutation of
two residues in this network (His81 and His217). The identities of both the substrate and the metal ion also
affect interactions within this H-bond network, thus leading to some mechanistic variations. Overall, the
mechanism employed by GpdQ is a paradigm of a substrate- and metal-ion-induced fit to optimize catalysis.
||The definitive version of this article was published in the Journal of the American Chemical Society, 2009, 131 (33), pp 11900–11908, DOI: 10.1021/ja903534f . Copyright © 2009 American Chemical Society
||Structural Flexibility; Reactivity; Bioremediator; Glycerophosphodiesterase; Mechanism; Hydrolysis; Enterobacter aerogenes (GpdQ);
||Science & Engineering > Chemistry
||29 May 2012 15:30
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||Journal of the American Chemical Society
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