Table 2.

Kinetic Parameters of Vv GT5 and Its R140W Mutant^a

Substrate	kcat (s−1)	Km (μM)	kcat/Km (s−1 μM−1)
Wild-Type Vv GT5 (GAT Activity)
Quercetinb	7.16 ± 0.37	5.60 ± 1.76	1.280
Quercetinc	1.63 ± 0.26	54.7 ± 17.4	0.030
Kaempferolb	6.05 ± 0.05	10.8 ± 0.4	0.560
Isorhamnetinb	0.413 ± 0.003	7.92 ± 0.32	0.052
UDP-GAb,d		17.7 ± 2.9	0.405
Vv GT5-R140W (GAT Activity)b
Quercetinb	0.00016 ± 0.00002	29.0 ± 15.5	5.52 × 10−6
UDP-GAe			0.0015 × 10−6
Vv GT5-R140W (GlcT activity)b
Quercetinb	2.94 ± 0.11	9.69 ± 1.50	0.303
Kaempferolb	1.04 ± 0.01	1.96 ± 0.17	0.531
Isorhamnetinb	0.148 ± 0.004	5.85 ± 0.86	0.025
UDP-Glcd		77.2 ± 8.4	0.038
Vv GT5-R140W (GalT activity)b
Quercetinb	0.429 ± 0.020	10.6 ± 1.5	0.041
Kaempferolb	0.395 ± 0.005	3.02 ± 0.19	0.131
Isorhamnetinb	0.0243 ± 0.0007	11.9 ± 0.9	0.002
UDP-Gald		225 ± 21	0.002

^aOnly flavonols were accepted as sugar acceptor substrates.

^bKinetic parameters were determined at pH 9.1 with the appropriate UDP-sugar (1.0 mM), as described in Methods.

^cKinetic parameters were determined at pH 7.0, as described in Methods.

^d100 μM quercetin was used as the sugar acceptor.

^eK_m value was too large to be determined precisely.