Reference : Study of the active site residues of a glycoside hydrolase family 8 xylanase
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/15408
Study of the active site residues of a glycoside hydrolase family 8 xylanase
English
Collins, T. [> > > >]
De Vos, D. [> > > >]
Hoyoux, A. [> > > >]
Savvides, S. N. [> > > >]
Gerday, Charles mailto [Université de Liège - ULg > Services généraux (Faculté des sciences) > Relations académiques et scientifiques (Sciences) >]
Van Beeumen, J. [> > > >]
Feller, Georges mailto [Université de Liège - ULg > Département des sciences de la vie > Labo de biochimie >]
25-Nov-2005
Journal of Molecular Biology
Academic Press Ltd Elsevier Science Ltd
354
2
425-435
Yes (verified by ORBi)
International
0022-2836
London
[en] catalytic site residues ; glycoside hydrolase family 8 ; xylanase
[en] Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k(cat) values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family. (c) 2005 Elsevier Ltd. All rights reserved.
http://hdl.handle.net/2268/15408
10.1016/j.jmb.2005.09.064

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