Abstract
The determination of enzyme kinetic parameters, such as the Km and kcat values, is an essential part of the characterization of newly discovered enzymes. This protocol describes the determination of enzyme kinetic parameters of the Barbarea vulgaris UDP-glycosyltransferases (UGTs) UGT73C11 and UGT73C13 toward the sapogenins oleanolic acid and hederagenin as sugar acceptor substrates. UGTs catalyze the transfer of glycosyl residues. They generally use uridine sugar nucleotides as their sugar donor substrates, whereas sugar acceptor substrates arise from structurally diverse sets of metabolite classes. This protocol is based on the quantification of 14C-labeled glycosides following thin layer chromatography (TLC)-based separation. The dependence of the measured signal on a universal radioactively-labeled sugar donor substrate allows the potential application of the protocol in combination with a wide range of different sugar acceptor substrates. However, since the here described TLC separation procedure has been optimized for the separation of sapogenins and their glycosides, some modifications may become necessary when investigating other compound classes.Figure 1. Glucosylation reaction catalyzed by UGT73C10-UGT73C13 from Barbarea vulgaris (Augustin et al., 2012). All four enzymes utilize uridine diphosphate glucose (UDP-glc) as glucosyl-moiety donor substrate and different sapogenins such as the oleanane sapogenins oleanolic acid and hederagenin as glucosyl-moiety acceptor substrates.
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Acknowledgments
This protocol was adapted and modified from various, previous 14C-UDP-glucose-based UGT enzyme assay protocols commonly applied in the Section for Plant Biochemistry – Department for Plant Biochemistry Biotechnology – Faculty of Life Sciences – University of Copenhagen and preceding organizations. This work was supported by the Danish Council for Independent Research, Technology, and Production Sciences (grant nos. 09–065899/FTP and 274–06–0370), by the Villum Kann Rasmussen Foundation to Pro-Active Plants, and by a PhD stipend from the Faculty of Life Sciences, University of Copenhagen (to J.M.A.).
References
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