title: Effect of the label of oligosaccharide acceptors on the kinetic parameters of nasturtium seed xyloglucan endotransglycosylase (XET).
abstract: Fluorescently labeled derivatives of a xyloglucan (XG) nonasaccharide Glc(4)Xyl(3)Gal(2) (XLLG) were used as glycosyl acceptors in assays of xyloglucan endotransglycosylase (XET) from germinated nasturtium (Tropaeolum majus) seeds. We have investigated how the type of the oligosaccharide label influences the kinetic parameters of the reaction. The fluorescent probes used to label XLLG were anthranilic acid (AA), 8-aminonaphtalene-1,3,6-trisulfonic acid (ANTS), fluorescein isothiocyanate (FITC), and sulforhodamine (SR), respectively. The obtained data were compared with those of the reactions where aldose and/or alditol forms of tritium-labeled xyloglucan-derived nonasaccharide served as the respective acceptors. Modification at C-1 of the reducing-end glucose in XLLG by substitution with the fluorophore markedly affected the kinetic parameters of the reaction. The Michaelis constants K(m) for individual acceptors increased in the order [1-(3)H]XLLG<XLLG-SR<[1-(3)H]XLLGol<XLLG-FITC<XLLG-ANTS<XLLG-AA, while the turnover numbers characterized by k(cat) decreased in the order XLLG-FITC>XLLG-SR>XLLG-ANTS>[1-(3)H]XLLGol>[1-(3)H]XLLG>XLLG-AA. Catalytic efficiency (expressed as k(cat)/K(m)) with XLLG labeled with SR or FITC was 15 and 28 times, respectively, higher than with the tritium-labeled natural substrate [1-(3)H]XLLG. Comparison of the kinetic parameters found with acceptors labeled with different types of labels enables to select the most effective substrates for the high-throughput assays of XET.
authors.affiliation: Institute of Chemistry, Centre for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, 84538 Bratislava, Slovakia.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Kosík
authors.forename: Soňa
authors.initials: S
authors.lastname: Garajová
authors.forename: Mária
authors.initials: M
authors.lastname: Matulová
authors.forename: Pavel
authors.initials: P
authors.lastname: Rehulka
authors.forename: Eva
authors.initials: E
authors.lastname: Stratilová
authors.forename: Vladimír
authors.initials: V
authors.lastname: Farkaš
mesh_terms: D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D005936:Glucans / Q000737:chemistry*; D016695:Glycosyltransferases / Q000737:chemistry*; D007700:Kinetics; D009844:Oligosaccharides / Q000737:chemistry*; D012639:Seeds / Q000201:enzymology*; D032388:Tropaeolum / Q000201:enzymology*; D014990:Xylans / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D005936:Glucans; D009844:Oligosaccharides; D014990:Xylans; C029353:xyloglucan; D016695:Glycosyltransferases; C073693:xyloglucan endotransglycosylase