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J. Biol. Chem., Vol. 283, Issue 32, 21864-21872, August 8, 2008

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Mechanism-based Labeling Defines the Free Energy Change for Formation of the Covalent Glycosyl-enzyme Intermediate in a Xyloglucan endo-Transglycosylase^*

Kathleen Piens¹², Régis Fauré¹, Gustav Sundqvist, Martin J. Baumann³, Marc Saura-Valls^¶, Tuula T. Teeri, Sylvain Cottaz, Antoni Planas^¶, Hugues Driguez, and Harry Brumer⁴

From the School of Biotechnology, Royal Institute of Technology, AlbaNova University Centre, SE-106 91 Stockholm, Sweden, the Centre de Recherche sur les Macromolécules Végétales, CNRS, BP53, 38041 Grenoble Cedex 9, France, and the ^¶Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain

Xyloglucan endo-transglycosylases (XETs) are key enzymes involved in the restructuring of plant cell walls during morphogenesis. As members of glycoside hydrolase family 16 (GH16), XETs are predicted to employ the canonical retaining mechanism of glycosyl transfer involving a covalent glycosyl-enzyme intermediate. Here, we report the accumulation and direct observation of such intermediates of PttXET16–34 from hybrid aspen by electrospray mass spectrometry in combination with synthetic "blocked" substrates, which function as glycosyl donors but are incapable of acting as glycosyl acceptors. Thus, GalGXXXGGG and GalGXXXGXXXG react with the wild-type enzyme to yield relatively stable, kinetically competent, covalent GalG-enzyme and GalGXXXG-enzyme complexes, respectively (Gal = Galβ(14), G = Glcβ(14), and X = Xyl(16)Glcβ(14)). Quantitation of ratios of protein and saccharide species at pseudo-equilibrium allowed us to estimate the free energy change (G⁰) for the formation of the covalent GalGXXXG-enzyme as 6.3–8.5 kJ/mol (1.5–2.0 kcal/mol). The data indicate that the free energy of the β(14) glucosidic bond in xyloglucans is preserved in the glycosyl-enzyme intermediate and harnessed for religation of the polysaccharide in vivo.

Received for publication, April 22, 2008 , and in revised form, May 28, 2008.

^* This work was supported in part by European Union Contract QLK5-CT-2001-00443, Grant 2005SGR 00883 from the Generalitat de Catalunya, and CNRS. The Centre de Recherche sur les Macromolécules Végétales is affiliated with University Joseph Fourier and is a member of the Institut de Chimie Moléculaire de Grenoble, FR-CNRS 2607. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Recipient of a Marie Curie postdoctoral fellowship from the European Union.

³ Recipient of predoctoral support from the Royal Institute of Technology Biofiber Materials Center.

⁴ Fellow (Rådsforskare) of the Swedish Research Council. To whom correspondence should be addressed. E-mail: harry{at}biotech.kth.se.

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