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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

TitleMechanism-based labeling defines the free energy change for formation of the covalent glycosyl-enzyme intermediate in a xyloglucan endo-transglycosylase
Publication TypeJournal Article
Year of Publication2008
AuthorsPiens, K, Faure, R, Sundqvist, G, Baumann, MJ, Saura-Valls, M, Teeri, TT, Cottaz, S, Planas, A, Driguez, H, Brumer, H
JournalJOURNAL OF BIOLOGICAL CHEMISTRY
Volume283
Pagination21864-21872
Date PublishedAUG 8
Type of ArticleArticle
ISSN0021-9258
Abstract

{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 = Gal beta(1 -> 4)

DOI10.1074/jbc.M803057200