Kinetic parameters of wild-type and mutants CYP24A1 for 1α,25(OH)2D3
The reaction was performed in the presence of 0.1 μM adrenodoxin, 0.01 μM ADR as described under Materials and Methods. Under these conditions, the sum of 23(S)-hydroxylated (M4) and 24(R)-hydroxylated (M5) products is more than 90% of total metabolites.
| CYP24A1 | Type | Km | kcat | kcat/Km |
|---|---|---|---|---|
| μM | min-1 | |||
| Rat WT | R | 0.19 ± 0.08 | 0.26 ± 0.10 | 1.4 ± 0.1 |
| Human WT | H | 0.35 ± 0.02 | 0.43 ± 0.04 | 1.2 ± 0.1 |
| T416A | R | 0.24 ± 0.10 | 0.25 ± 0.07 | 1.2 ± 0.3 |
| T416S | R | 0.22 ± 0.01 | 0.30 ± 0.02 | 1.4 ± 0.1 |
| T416V | H | 1.04 ± 0.32 | 0.72 ± 0.31 | 0.71 ± 0.31 |
| T416M | H | 0.66 ± 0.06 | 0.21 ± 0.09 | 0.32 ± 0.15 |
| I500T | H | 0.14 ± 0.04 | 0.12 ± 0.03 | 0.92 ± 0.07 |
| I500L | H | 0.30 ± 0.12 | 0.05 ± 0.01 | 0.17 ± 0.03 |
| I500F | N | 1.14 ± 0.40 | 0.11 ± 0.04 | 0.10 ± 0.04 |
R, H, and N indicate rat-, human-, and novel-type metabolism, respectively.