Table 2. Apparent Kinetic Parameters for FIH and Its Variantsa.
| kcat (min–1)b | kcat/KM(CTAD) (μM–1 min–1)b | KM(CTAD) (μM)b | KD(CTAD) (μM)c | KM(αKG) (μM)d | |
|---|---|---|---|---|---|
| WT | 30 ± 2.5e | 0.4 ± 0.1e | 70 ± 20e | 78 ± 7f | 16 ± 3.0 |
| Q239A | 1.3 ± 0.10 | 0.021 ± 0.002 | 61 ± 10 | 100 ± 16 | 5.0 ± 0.5 |
| Q239N | 0.14 ± 0.02 | 2.0 × 10–3 ± 8 × 10–4 | 74 ± 30 | 98 ± 10 | 4.0 ± 0.4 |
| Q239H | 0.023 ± 0.003 | (3.4 ± 1) × 10–4 | 68 ± 18 | 64 ± 14 | 7.0 ± 1.4 |
| Q239E | 0.024 ± 0.002 | 3.4 × 10–4 ± 7 × 10–5 | 71 ± 10 | 75 ± 15 | 4.7 ± 2.0 |
| Q239L | <0.005g | <8 × 10–5g | NDh | 80 ± 8 | NDh |
a
In 50 mM HEPES (pH 7.00) at 37.0 °C.
b
Assays in which CTAD was the varied substrate, in ascorbate (2 mM), αKG (500 μM), FeSO4 (25 μM), and CTAD (0–300 μM).
c
Determined using intrinsic tryptophan fluorescence with Co-substituted enzyme.
d
Assays in which αKG was the varied substrate, in ascorbate (2 mM), αKG (2–200 μM), FeSO4 (25 μM), and CTAD (100 μM).
e
From ref (25).
f
From ref (30).
g
No activity detected; estimated detection limits as reported.
h
Not determined.