Mechanism-based labeling defines the free energy change for formation of the covalent glycosyl-enzyme intermediate in a xyloglucan endo-transglycosylase
2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 32, 21864-21872 p.Article in journal (Refereed) Published
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), G = Glc beta(1 -> 4), and X = Xyl alpha(1 -> 6) Glc beta(1 -> 4)). Quantitation of ratios of protein and saccharide species at pseudo-equilibrium allowed us to estimate the free energy change (Delta G(0)) 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 beta(1 -> 4) glucosidic bond in xyloglucans is preserved in the glycosyl-enzyme intermediate and harnessed for religation of the polysaccharide in vivo.
Place, publisher, year, edition, pages
2008. Vol. 283, no 32, 21864-21872 p.
Biomaterials, Free energy, Gene transfer, High performance liquid chromatography, Mass spectrometry, Plant cell culture, Plants (botany), Polysaccharides, Sugars, Direct observations, Electrospray mass spectrometries, Enzyme complexes, Free energy changes, Glucosidic bonds, Glycoside hydrolase family 16, Glycosyl, Glycosyl acceptors, Glycosyl donors, In-vivo, Key enzymes, Plant cell walls, Transglycosylases, Xyloglucan, Xyloglucans
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-8351DOI: 10.1074/jbc.M803057200ISI: 000258114700005ScopusID: 2-s2.0-52049094160OAI: oai:DiVA.org:kth-8351DiVA: diva2:13649
QC 20100913. Uppdaterad från manuskript till artikel (20100913).2008-05-072008-05-072010-09-13Bibliographically approved