Table 1.

Steady-state kinetic parameters of WT-FIH, D201G and D201E^a

	kcatapp (min−1) b	kcat/KM(CTAD)app (μM−1 min−1) b	KM(CTAD)app (μM) b	KM(αKG)app (μM) c
WT FIH	33 ± 2	0.42 ± 0.08	78 ± 17	20 ± 5
D201E	1.6 ± 0.2	0.022 ± 0.002	70 ± 15	26 ± 8
D201G	23 ± 2	0.34 ± 0.05	71 ± 12	23 ± 6
D201A	d	d	d	d

^aBased on K_M(O2), each variant had a different degree of O₂ saturation. WT, 52%; D201E, 75%; D201G, 65%.

^bReactions contained ascorbate (2 mM), αKG (100 μM), FeSO₄ (25 μM) and CTAD (0–260 μM) in 50 mM HEPES containing 100 mM NaCl, pH 7.00, 37.0 °C.

^cReactions contained ascorbate (2 mM), αKG (0–200 μM), FeSO₄ (25 μM) and CTAD (80 μM) in 50 mM HEPES containing 100 mM NaCl, pH 7.00, 37.0 °C.

^dNo hydroxylation activity detected; Estimated hydroxylation detection limit <0.005 min⁻¹, if active.