Kinetic analysis of UGT1A6 control and 6H371A and 6E379A mutant forms
Constant UDPGA concentration (5 or 20 mM). 1-Naphthol and 4-MU kinetics was measured with eight aglycone concentrations (1-naphthol between 1 and 200 μM and 4-MU between 10 and 1500 μM and 5 mM UDPGA). The scopoletin kinetics was measured with nine different concentrations between 25 and 4000 μM and 20 mM UDPGA. Numbers in parentheses indicate the ratio Km(mutant)/Km(control) (i.e., to indicate how much the mutant Km value increased).
UGT & Substrate | Km | Vmax Normalized | Vmax |
|---|---|---|---|
| μM | pmol/min/mg | % of control | |
| UGT1A6 | |||
| Scopoletin | 330 ± 20 (1) | 9320 ± 580 | 100 |
| 1-Naphthol | 2.7 ± 0.3 (1) | 2130 ± 130 | 100 ± 2.0 |
| 4-MU | 170 ± 11 (1) | 7840 ± 490 | 100 ± 1.7 |
| 6H371A | |||
| Scopoletin | 1710 ± 70 (5) | 1430 ± 90 | 15 ± 1.4 |
| 1-Naphthol | 83 ± 5 (31) | 330 ± 20 | 15 ± 1.4 |
| 4-MU | 660 ± 30 (4) | 100 ± 6 | 1.3 ± 0.1 |
| 6E379A | |||
| Scopoletin | 1470 ± 80 (4) | 1340 ± 110 | 14 ± 1.5 |
| 1-Naphthol | 48 ± 2 (18) | 600 ± 50 | 28 ± 2.9 |
| 4-MU | 580 ± 70 (3) | 180 ± 20 | 2.3 ± 0.3 |