Kinetic Investigations of the Role of Factor Inhibiting Hypoxia-inducible Factor (FIH) as an Oxygen Sensor

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-alpha, 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 O-2 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 O-2 concentration and slow reaction with O-2. To ascertain whether these parameters are conserved among the HIF hydroxylases, we compared the reactions of FIH and PHD2 with O-2. Consistent with previous reports, we found lower K-m(app)(O-2) values for FIH than for PHD2 with all HIF-derived substrates. Under pre-steady-state conditions, the O-2-initiated FIH reaction is significantly faster than that of PHD2. We then investigated the kinetics with respect to O-2 of the FIH reaction with ankyrin repeat domain (ARD) substrates. FIH has lower K-m(app)(O-2) values for the tested ARDs than HIF-alpha substrates, and pre-steady-state O-2-initiated reactions were faster with ARDs than with HIF-alpha substrates. The results correlate with cellular studies showing that FIH is active at lower O-2 concentrations than the PHDs and suggest that competition between HIF-alpha 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.