Kinetic Investigations of the Role of Factor Inhibiting Hypoxia-inducible Factor (FIH) as an Oxygen Sensor.
Bottom Line: FIH is an asparaginyl hydroxylase catalyzing post-translational modification of HIF-α, resulting in reduction of HIF-mediated transcription.Consistent with previous reports, we found lower Km(app)(O2) values for FIH than for PHD2 with all HIF-derived 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.
Affiliation: From the Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom and.Show MeSH
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Mentions: To further investigate the difference in the O2 dependence of the FIH-catalyzed hydroxylation of HIF-α and ARD substrates, stopped-flow and rapid quench-flow experiments were performed. Interestingly, the determined pre-steady-state kinetic parameters suggested markedly faster O2-initiated reaction rates in the presence of ankyrin rather than HIF-α CAD substrates. Upon reaction of FIH·Fe(II)·2OG·1CA20-mer and FIH·Fe(II)·2OG·tnkrs-120-mer complexes with O2, rate constants of product accumulation were determined to be 1.3 ± 0.2 and 1.5 ± 0.1 s−1, respectively (i.e. ∼10-fold faster than the reaction with HIF 35-mer substrates) (Fig. 9). Rates of succinate production were also determined and showed similar relative changes (determined rate constants are summarized in Table 5 and supplemental Fig. S6); for both HIF-α isoforms as well as ankyrin substrates, the reaction was fully coupled. In previously reported steady-state kinetic studies (43), when the length of the peptide substrate was increased from 20-mer to 34-mer for ankyrin peptides, a ∼100-fold increase in substrate affinity was observed. O2 initiation of the FIH reaction with a 35-mer tankyrase-1 peptide resulted in product formation at a rate ∼7-fold faster than for the 20-mer (determined rate constants 1.5 ± 0.1 s−1 for tnkrs-120-mer hydroxylation, 11 ± 3 s−1 for tnkrs-135-mer hydroxylation). Consistent with faster hydroxylation, stopped-flow UV-visible absorbance experiments revealed that an absorbance minimum at 500 nm was reached faster for ankyrin substrates compared with HIF-α substrates (Fig. 10A). As for FIH-catalyzed HIF-α CAD hydroxylation, a transient species absorbing at 310 nm was observed subsequent to FIH-catalyzed ARD hydroxylation (Fig. 10B) (i.e. accumulation of an Fe(IV)-oxo intermediate was not observed (at 320 nm) in FIH catalysis).
Affiliation: From the Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom and.