Table 3. Km and Vmax values for CYP3A4 and CYP2B6 enzymes with different mutants of POR were determined by their specific substrates testosterone and bupropion.
| POR | CYP3A4 - Testosterone | CYP2B6 – Bupropion | |||||||
| Km (nM/min/pmol) | Vmax (% of WT) | Clint (μM) | Km (nM/min/pmol) | Vmax (% of WT) | Clint (μM) | ||||
| WT | 236.7±10.3 | 13.70±0.88 | 0.058±0.0039 (100) | 388.8±20.02 | 10.59±0.54 | 0.027±0.0027 (100) | |||
| K49N | 197.7±9.0* | 15.08±0.31 | 0.076±0.0040 (131)* | 311.1±12.13* | 6.12±0.43* | 0.020±0.0017 (74) # | |||
| A115V | 211.8±13.8 | 16.65±0.53* | 0.079±0.0058 (136)* | 294.6±26.19* | 7.44±0.30* | 0.025±0.0024 (93) | |||
| Y181D | – | – | – | 369.6.±10.96 | 7.10±0.26* | 0.019±0.0015 (70) # | |||
| S244C | 219.7±22.6 | 14.65±0.74 | 0.067±0.0091 (115) | 316.5±13.37* | 9.95±0.44 | 0.031±0.0018 (115) | |||
| A287P | – | – | – | 475.1±24.91* | 9.28±0.65 | 0.020±0.0015 (74) # | |||
| G413S | 183.3±13.3* | 17.57±0.58* | 0.096±0.0044(165)* | 299.7±13.07* | 6.65±0.36* | 0.022±0.0019 (81) | |||
The ratio of Vmax to Km was used as an index of catalytic efficiency; the activity of each POR mutant co-expressed with CYPs was calculated and expressed as a percentage of the activity of wild-type POR, arbitrarily set at 100%.
The data were represented as mean ± S.D. of three independent experiments.
CLint = Vmax/Km.
*
: p<0.01 in comparison with the cells expressing wild-type (WT) POR.
#
: p<0.05 in comparison with the cells expressing wild-type (WT) POR.
Dash (–) indicates not detectable.