TABLE 5.
Pre-steady-state kinetic parameters of FIH-catalyzed ARD hydroxylation
Conditions were as follows. 0.5 mm apo-FIH, 0.4 mm Fe(II), 5 mm 2OG, and 1 mm substrate in 50 mm HEPES (pH 7.5) were rapidly mixed with O2-saturated buffer at 5 °C in a 1:1 ratio. ND, not determined.
| Substrate | Stopped-flow, 500 nm |
Rapid quench-flow |
||
|---|---|---|---|---|
| kfall phase | krise phase | kproduct | ksuccinate | |
| s−1 | s−1 | s−1 | s−1 | |
| 1CA | 17.6 ± 0.1 | 0.4 ± 0.1 | 1.3 ± 0.2 | 0.8 ± 0.3 |
| 2CA | 11.9 ± 0.2 | 0.6 ± 0.1 | 0.5 ± 0.1 | ND |
| 3CA | 10.5 ± 0.1 | 1.7 ± 0.3 | 0.051 ± 0.001 | ND |
| tnkrs-120-mer | 16.7 ± 0.2 | 0.4 ± 0.2 | 1.5 ± 0.1 | 1.0 ± 0.3 |
| tnkrs-135-mer | 14.8 ± 0.2 | 0.27 ± 0.03 | 11 ± 3 | 10 ± 3 |