Table 1.
Steady state kinetics of WT-PHD2 and variants with varied [2OG].a
| Enzyme |
kcat min−1 |
kcat/KM(2OG) µM−1min−1 |
KI(2OG) µM |
KM(2OG), µM |
KD(2OG)b µM |
|---|---|---|---|---|---|
| WT-PHD2 | 2.3 ± 0.1 | 2.7 ± 0.4 | >2.6×104 | 0.9 ± 0.2 | 0.7 ± 0.2 |
| Thr387→Ala | 32.9 ± 1.4 | 2.7 ± 0.3 | 2150 ± 340 | 12.0 ± 1.6 | 1.2 ± 0.2 |
| Thr387→Asn | 1.0 ± 0.1 | 1.5 ± 0.2 | >5×104 | 0.7 ± 0.1 | 0.6 ± 0.2 |
a
. Reactions contained (NH4)2Fe(SO4)2 (10 µM), ascorbic acid (1 mM), 2OG (1–3000 µM), and CODD (10 µM) in 50 mM HEPES pH 7.00, 37°C, ambient [O2](217 µM).
b
. Fluorescence titrations: PHD2 (1.1 µM), MnSO4 (20 µM) in 50 mM HEPES pH 7.00 titrated with 2OG (500 µM).