Effect of the label of oligosaccharide acceptors on the kinetic parameters of nasturtium seed xyloglucan endotransglycosylase (XET)

Fluorescently labeled derivatives of a xyloglucan (XG) nonasaccharide Glc4Xyl3Gal2 (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 Km for individual acceptors increased in the order [1-3H]XLLG < XLLG-SR < [1-3H]XLLGol < XLLG-FITC < XLLG-ANTS < XLLG-AA, while the turnover numbers characterized by kcat decreased in the order XLLG-FITC > XLLG-SR > XLLG-ANTS > [1-3H]XLLGol > [1-3H]XLLG > XLLG-AA. Catalytic efficiency (expressed as kcat/Km) with XLLG labeled with SR or FITC was 15 and 28 times, respectively, higher than with the tritium-labeled natural substrate [1-3H]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.