Table 1.
Effects of mutations of candidate sites for phosphorylation of P450c17
| Mutation | Hydroxylase activity of wt (%) | Lyase activity of wt without b5 (%) | Lyase activity of wt with b5 (%) |
|---|---|---|---|
| WT | 100 (20 ± 0.73) | 100 (8.9 ± 2.3) | 100 (22 ± 7.9) |
| S65D | 9 | 49 | 18 |
| T70E | 88 | 114 | 67 |
| T72E | 53 | 98 | 12 |
| S94D | 43 | 18 | 6 |
| S168D | 8 | 0 | 0 |
| S187D | 2 | 22 | 4 |
| S256D | 62 | 31 | 33 |
| S258D | 75 | 17 | 23 |
| S288D | 10 | 77 | 10 |
| T343E | 87 | 39 | 48 |
| S380D | 17 | 13 | 16 |
| S427D | 21 | 4 | 0 |
| S431D | 4 | 6 | 0 |
| S441D | 20 ± 8 (P < 0.0001)a | 0 (P = 0.0003)a | 4 |
| S94D/S234D | 66 ± 15 (P = 0.0116)a | 10 ± 2 (P = 0.0005)a | 13 |
| S94D/T265E | 31 ± 11 (P < 0.0001)a | 14 ± 4 (P = 0.0008)a | 3 |
| S94D/S309D | 0 | 27 | 3 |
| S94D/T314E | 43 ± 8 (P < 0.0001)a | 14 ± 4 (P = 0.0008)a | 2 |
| S94D/T343E | 110 | 0 | 0 |
| S94D/T356E | 6 | 21 | 12 |
| S94D/S379D | 1 ± 2 (P < 0.0001)a | 7 ± 2 (P = 0.0004)a | 1 |
| S94D/S380D | 0 | 19 | 7 |
| S94D/S427D | 5 | 0 | 0 |
| S94D/S429D | 6 ± 5 (P < 0.0001)a | 15 ± 5 (P = 0.0010)a | 2 |
| T341E/T343E | 67 | 9 | 44 |
| S256D/S258D/T260D | 8 | 0 | 0 |
| S94D/T341E/T343E | 5 | 6 | 2 |
The data are mean ± sd of quadruplicate experiments of hydroxylase and 17,20 lyase assays for constructs S441D, S94D/S234D, S94D/T265E, S94D/T314E, S94D/S379D, and S94D/S429D and duplicate experiments for the remaining constructs. The hydroxylase and lyase activities for the WT construct are given in parentheses as femtomoles per picomole P450c17 per minute (mean ± sd). WT, Wild type.
a
Significant differences in hydroxylase and lyase activities from WT for constructs S441D, S94D/S234D, S94D/T265E, S94D/T314E, S94D/S379D, and S94D/S429D.