title: Kinetic Investigations of the Role of Factor Inhibiting Hypoxia-inducible Factor (FIH) as an Oxygen Sensor.
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.
authors.affiliation: From the Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom and.
authors.forename: Hanna
authors.initials: H
authors.lastname: Tarhonskaya
authors.affiliation: From the Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom and.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Hardy
authors.affiliation: From the Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom and.
authors.forename: Emily A
authors.initials: EA
authors.lastname: Howe
authors.affiliation: From the Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom and.
authors.forename: Nikita D
authors.initials: ND
authors.lastname: Loik
authors.affiliation: the OXION Proteomics Facility, Department of Physiology, Anatomy, and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, United Kingdom.
authors.forename: Holger B
authors.initials: HB
authors.lastname: Kramer
authors.affiliation: From the Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom and.
authors.forename: James S O
authors.initials: JS
authors.lastname: McCullagh
authors.affiliation: From the Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom and.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.affiliation: From the Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom and emily.flashman@chem.ox.ac.uk.
authors.forename: Emily
authors.initials: E
authors.lastname: Flashman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014158:Transcription, Genetic
chemical_list: D017487:Ankyrins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011994:Recombinant Proteins; D012097:Repressor Proteins; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human; C490190:EGLN1 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D010100:Oxygen
keywords: HIF hydroxylase; ankyrin repeat domain; dioxygenase; factor inhibiting HIF; hypoxia; hypoxia-inducible factor (HIF); kinetics; oxygen sensor; pre-steady-state kinetics; prolyl hydroxylase