The determination of enzyme kinetic parameters, such as the K_m and k_cat 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.