Abstract
The hypoxia-inducible factor (HIF) hydroxylases regulate hypoxia sensing in animals. In humans, they comprise three prolyl hydroxylases (PHD1-3 or EGLN1-3) and factor inhibiting HIF (FIH). FIH is an asparaginyl hydroxylase catalyzing post-translational modification of HIF-α, resulting in reduction of HIF-mediated transcription. Like the PHDs, FIH is proposed to have a hypoxia-sensing role in cells, enabling responses to changes in cellular O2 availability. PHD2, the most important human PHD isoform, is proposed to be biochemically/kinetically suited as a hypoxia sensor due to its relatively high sensitivity to changes in O2 concentration and slow reaction with O2. To ascertain whether these parameters are conserved among the HIF hydroxylases, we compared the reactions of FIH and PHD2 with O2. Consistent with previous reports, we found lower Km(app)(O2) values for FIH than for PHD2 with all HIF-derived substrates. Under pre-steady-state conditions, the O2-initiated FIH reaction is significantly faster than that of PHD2. We then investigated the kinetics with respect to O2 of the FIH reaction with ankyrin repeat domain (ARD) substrates. FIH has lower Km(app)(O2) values for the tested ARDs than HIF-α substrates, and pre-steady-state O2-initiated reactions were faster with ARDs than with HIF-α substrates. The results correlate with cellular studies showing that FIH is active at lower O2 concentrations than the PHDs and suggest that competition between HIF-α and ARDs for FIH is likely to be biologically relevant, particularly in hypoxic conditions. The overall results are consistent with the proposal that the kinetic properties of individual oxygenases reflect their biological capacity to act as hypoxia sensors.
Highlights
The hypoxia-inducible factor (HIF) hydroxylases (FIH and PHDs) regulate hypoxia sensing in animals
Differences were observed in the relative efficiencies of catalysis in overall time course assays; consistent with a previous report [46], PHD2 showed a higher level of activity toward CODD (70 –90% substrate hydroxylation after 15 min for both HIF-1␣ and HIF-2␣, respectively) than NODD (50 and 20% substrate hydroxylation for HIF-1␣ and HIF-2␣, respectively) (Fig. 3, A and B)
Increasing the length of the HIF-1␣ C-terminal transactivation domain (CAD) peptide resulted in nearly complete substrate hydroxylation, whereas analyses increasing the length of the HIF-2␣ CAD showed only a 10% increase in activity and Ͻ30% substrate hydroxylation (Fig. 3, C and D)
Summary
The hypoxia-inducible factor (HIF) hydroxylases (FIH and PHDs) regulate hypoxia sensing in animals. Significance: We provide a kinetic rationale for cellular observations that FIH activity is more hypoxia-tolerant than that of the PHDs. The hypoxia-inducible factor (HIF) hydroxylases regulate hypoxia sensing in animals. PHD2, the most important human PHD isoform, is proposed to be biochemically/kinetically suited as a hypoxia sensor due to its relatively high sensitivity to changes in O2 concentration and slow reaction with O2. To ascertain whether these parameters are conserved among the HIF hydroxylases, we compared the reactions of FIH and PHD2 with O2. The work demonstrates that, at least for PHD2 and FIH, the kinetic properties of these enzymes with respect to O2 reflect their cellular hypoxia-sensing ability
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