Table I.

Kinetic parameters for wild-type BpUGT94B1 and point mutants

Sugar donor kinetic parameters were determined using the acceptor cyanidin 3-O-glucoside as substrate. Sugar acceptor kinetic parameters were determined using the sugar donor UDP-GlcUA as substrate. ND, Not determined. Kinetic parameters and their ses were calculated by fitting the initial velocity data to the Michaelis-Menten equation by nonlinear regression analysis. Experimental data are presented in Figures 7, 8, and 9. For determination of relative Kcat product was quantified relative to the amount of product formed by the wild-type enzyme at 10 mm UDP-GlcUA and 25 mm UDP-Glc, respectively.

EnzymesubstrateKm ± sdRelative kcat ± sdRelative kcat/Km
mmmin−1min−1 mm−1
Wild typeUDP-GlcUA1.1 ± 0.1100 ± 3.990.9
R25SUDP-GlcUA1.4 ± 0.32.5 ± 0.21.8
R25GUDP-GlcUA2.1 ± 0.30.6 ± 0.030.29
R25KUDP-GlcUA7.0 ± 0.51.6 ± 0.050.23
N123AUDP-GlcUA1.6 ± 0.2ND
L148AUDP-GlcUA1.9 ± 0.1ND
D152AUDP-GlcUA2.7 ± 0.3ND
I187AUDP-GlcUA1.0 ± 0.2ND
Wild typeUDP-Glc7.3 ± 0.9100 ± 513.7
R25SUDP-Glc2.9 ± 0.5218 ± 1475.2
Wild typecyanidin 3-O-glucoside0.8 ± 0.2ND