TABLE 2.

Steady-state kinetic parameters of FIH and PHD2 catalysis

Conditions were as follows. 0.1–5 μm FIH, peptide substrate (varied), 50 μm Fe(II), 2OG (varied), and 1 mm l-ascorbate in 50 mm HEPES (pH 7.5) were incubated at 37 °C. 4 μm PHD2, peptide substrate (varied), 50 μm Fe(II), 2OG (varied), and 4 mm l-ascorbate in 50 mm HEPES (pH 7.5) were incubated at 37 °C. Hydroxylation levels were analyzed by MALDI-TOF-MS. The dependences of the initial rate on the substrate concentration are presented in supplemental Figs. S1–S3.

Enzyme	Substrate	Kmapp(2OG)	Kmapp(peptide)	Kmapp(O2)	kcatapp
		μm	μm	μm	s−1
FIH	HIF-1α CAD35-mer	110 ± 20	180 ± 30	110 ± 30	0.56 ± 0.04
FIH	HIF-2α CAD35-mer	19 ± 6a	315 ± 40a	110 ± 10	0.049 ± 0.005
PHD2	HIF-1α CODD	13 ± 2	10 ± 6	460 ± 30	0.060 ± 0.006
PHD2	HIF-1α NODD	30 ± 9	11 ± 2	>450	0.028 ± 0.001
PHD2	HIF-2α CODD	9 ± 2	34 ± 10	>450	0.069 ± 0.006
PHD2	HIF-2α NODD	17 ± 5	50 ± 8	410 ± 80	0.013 ± 0.001

^a Substrate inhibition at 2OG concentrations >100 μm and HIF-2α CAD_35-mer concentrations >700 μm was observed.