Table II.
Catalytic parameters of the reactions catalyzed by CYP76B1 in the presence of different P450 reductases
| Activity | Coexpressed Reductase | Km | kcat | kcat/Km |
|---|---|---|---|---|
| μm | min−1 | min−1 μm−1 | ||
| MCOD | Yeast | 899 | 13 | 0.01 |
| ATR1 | 808 | 31 | 0.04 | |
| ECOD | Yeast | 74 | 10 | 0.13 |
| ATR1 | 60 | 30 | 0.5 | |
| MROD | Yeast | 0.23 | 15 | 64 |
| ATR1 | 0.17 | 41 | 237 | |
| EROD | Yeast | 0.49 | 6 | 12 |
| ATR1 | 0.17 | 28 | 165 | |
| IPUDM | Yeast | 361 | 40 | 0.11 |
| ATR1 | 289 | 147 | 0.51 | |
| CTUDM | Yeast | 202 | 376 | 1.8 |
| ATR1 | 179 | 803 | 4.5 | |
| Monodemethyl-CTUDM | ATR1 | 81 | 46 | 0.57 |
The CYP76B1 concentrations in the MCOD, ECOD, MROD and EROD fluorometric assays were 1.5 or 2.1 nm, depending on the coexpressed ATR1 or yeast reductases. In the CTUDM assays, the CYP76B1 concentrations were 1.7 nm (ATR1) and 8 nm (yeast), respectively. It was 12 nm for the determination of the catalytic parameters of monodemethyl-CTUDM, and 1.7 nm (ATR1) or 20 nm (yeast) in the IPUDM assays. Products were quantified by direct TLC analysis of 100 μL of the acidified incubation medium after 6 min of incubation at 30°C for the CTUDM and IPUDM assays, and after 9 min of incubation for the monodemethyl-CTUDM.