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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.
- Source :
-
Journal of Biological Chemistry . 8/8/2008, Vol. 283 Issue 32, p21864-21872. 9p. 2 Diagrams, 2 Charts, 6 Graphs. - Publication Year :
- 2008
-
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 = Galβ(1→4), G = Glcβ(1→4), and X = Xylα(1→6)Glcβ(1→4)). Quantitation of ratios of protein and saccharide species at pseudo-equilibrium allowed us to estimate the free energy change (ΔG0)tor 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 18(1→4) glucosidic bond in xyloglucans is preserved in the glycosyl-enzyme intermediate and harnessed for religation of the polysaccharide in vivo. [ABSTRACT FROM AUTHOR]
- Subjects :
- *ENZYMES
*GLYCOSYLTRANSFERASES
*PLANT cell walls
*MORPHOGENESIS
*GLYCOSIDASES
Subjects
Details
- Language :
- English
- ISSN :
- 00219258
- Volume :
- 283
- Issue :
- 32
- Database :
- Academic Search Index
- Journal :
- Journal of Biological Chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 34020671
- Full Text :
- https://doi.org/10.1074/jbc.M803057200