Table 3.
Kinetic Analysis of UGT3A2 Variants for UDP-Sugars Using 1-OH-Pyrenea
| UDP-glucose |
UDP-xylose |
UDP-glucose/UDP-xylose |
|||||
|---|---|---|---|---|---|---|---|
| UGT3A2 | Km (μM) |
Vmaxb (pmol·min−1·mg−1) |
Vmax/Km (μL·min−1·mg−1) |
Km (μM) |
Vmaxb (pmol·min−1·mg−1) |
Vmax/Km (μL·min−1·mg−1 |
Vmax/Km ratioc |
| WT | 214 ± 40 | 997 ± 131 | 4.7 ± 0.3 | 728 ± 169d | 992 ± 316 | 1.4 ± 0.4d | 3.4 |
| Y74N | 3359 ± 414 | 43 ± 7 | 0.013 ± 0.001 | NA | NA | NA | NC |
| D140N | 774 ± 5 | 253 ± 53 | 0.33 ± 0.07 | 1050 ± 517 | 321 ± 111 | 0.35 ± 0.11 | 0.94 |
| A344T | 597 ± 163 | 836 ± 84 | 1.5 ± 0.4 | 489 ± 26 | 455 ± 27d | 0.93 ± 0.01 | 1.6 |
| S435Y | 1284 ± 356 | 707 ± 233 | 0.55 ± 0.07 | 1708 ± 203 | 655 ± 217 | 0.47 ± 0.11 | 1.2 |
| A436T | 1008 ± 60 | 197 ± 16 | 0.20 ± 0.01 | 1072 ± 311 | 129 ± 38d | 0.13 ± 0.04d | 1.5 |
| R445C | 1322 ± 250 | 140 ± 14 | 0.11 ± 0.02 | 2946 ± 1334 | 159 ± 50d | 0.061 ± 0.018d | 1.8 |
| Y474C | 3133 ± 604 | 95 ± 13 | 0.032 ± 0.010 | >4000 | >24 | NC | NC |
a
Kinetic parameters of UGT3A2 variants for UDP-sugars were determined using 16 μM 1-OH-pyrene. Data are expressed as the mean ± SD of three independent experiments. Km, Vmax, Vmax/Km represent the mean ± SD of three independent experiments
b
Vmax was calculated per total microsomal protein, normalized to relative UGT3A2 expression.
c
Vmax/Km ratio shows the relative activity for UDP-glucose versus UDP-xylose.
d
P < 0.05 for UDP-xylose vs corresponding value for UDP-glucose. NC, not calculated because it did not reach saturation. NA, no activity.