TABLE 3.

Kinetic parameters of pre-steady-state PHD2- and FIH-catalyzed hydroxylation

Conditions were as follows: an anaerobically prepared mixture of 0.8 mm PHD2, 1 mm peptide substrate, 5 mm 2OG, and 0.7 mm Fe(II) or 0.5 mm FIH, 1 mm peptide substrate, 5 mm 2OG, and 0.4 mm Fe(II) in 50 mm HEPES (pH 7.5) was rapidly mixed with O₂-saturated buffer at 5 °C. ND, not determined.

Enzyme	Substrate	Stopped-flow, 520 nm (PHD2), 500 nm (FIH)		Rapid quench-flow
		kfall phase	krise phase	kproduct	ksuccinate
		s−1	s−1	s−1	s−1
PHD2	CODD HIF-1α	0.040 ± 0.007	0.015 ± 0.005	0.0155 ± 0.0012	0.0130 ± 0.0020
PHD2	CODD HIF-2α	0.450 ± 0.010	0.027 ± 0.001	0.0124 ± 0.0030	0.0120 ± 0.0070
PHD2	NODD HIF-1α	0.178 ± 0.006	0.002 ± 0.001	0.0080 ± 0.0020	0.0060 ± 0.0010
PHD2	NODD HIF-2α	0.195 ± 0.009	0.003 ± 0.001	0.0199 ± 0.0032	0.0270 ± 0.0060
FIH	CAD HIF-1α35-mer	4.5 ± 0.3	0.009 ± 0.005	0.33 ± 0.07	0.3 ± 0.1
FIH	CAD HIF-2α35-mer	4.9 ± 0.2	0.017 ± 0.01	0.12 ± 0.01	0.11 ± 0.03
FIH	CAD HIF-1α19-mer	ND	ND	0.013 ± 0.003	0.016 ± 0.004