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

Tarhonskaya H, Hardy AP, Howe EA, Loik ND, Kramer HB, McCullagh JS, Schofield CJ, Flashman E - J. Biol. Chem. (2015)

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.Under pre-steady-state conditions, the O2-initiated FIH reaction is significantly faster than that of PHD2.

View Article: PubMed Central - PubMed

Affiliation: From the Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom and.

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Stopped-flow UV-visible absorbance changes upon FIH-catalyzed hydroxylation of HIF-1/2α CAD and ARD substrates. Reaction mixtures containing anaerobic 0.5 mm apo-FIH, 0.4 mm Fe(II), 5 mm 2OG, and 1 mm peptide in HEPES 50 mm (pH 7.5) were rapidly mixed with O2-saturated buffer at 5 °C in 1:1 ratio and analyzed using a photodiode array detector. The data were fitted with a double exponential function. A, decay at 500 nm (representing enzyme·substrate complex depletion) observed upon FIH-catalyzed hydroxylation of ARD substrates compared with HIF-1/2α CAD. B, formation and degradation of 310 nm species observed upon FIH-catalyzed HIF-1/2α CAD and ARD hydroxylation. C, decay at 500 nm observed upon FIH-catalyzed hydroxylation of 1CA, 2CA, and 3CA substrates.
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Figure 10: Stopped-flow UV-visible absorbance changes upon FIH-catalyzed hydroxylation of HIF-1/2α CAD and ARD substrates. Reaction mixtures containing anaerobic 0.5 mm apo-FIH, 0.4 mm Fe(II), 5 mm 2OG, and 1 mm peptide in HEPES 50 mm (pH 7.5) were rapidly mixed with O2-saturated buffer at 5 °C in 1:1 ratio and analyzed using a photodiode array detector. The data were fitted with a double exponential function. A, decay at 500 nm (representing enzyme·substrate complex depletion) observed upon FIH-catalyzed hydroxylation of ARD substrates compared with HIF-1/2α CAD. B, formation and degradation of 310 nm species observed upon FIH-catalyzed HIF-1/2α CAD and ARD hydroxylation. C, decay at 500 nm observed upon FIH-catalyzed hydroxylation of 1CA, 2CA, and 3CA substrates.

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).


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

Tarhonskaya H, Hardy AP, Howe EA, Loik ND, Kramer HB, McCullagh JS, Schofield CJ, Flashman E - J. Biol. Chem. (2015)

Stopped-flow UV-visible absorbance changes upon FIH-catalyzed hydroxylation of HIF-1/2α CAD and ARD substrates. Reaction mixtures containing anaerobic 0.5 mm apo-FIH, 0.4 mm Fe(II), 5 mm 2OG, and 1 mm peptide in HEPES 50 mm (pH 7.5) were rapidly mixed with O2-saturated buffer at 5 °C in 1:1 ratio and analyzed using a photodiode array detector. The data were fitted with a double exponential function. A, decay at 500 nm (representing enzyme·substrate complex depletion) observed upon FIH-catalyzed hydroxylation of ARD substrates compared with HIF-1/2α CAD. B, formation and degradation of 310 nm species observed upon FIH-catalyzed HIF-1/2α CAD and ARD hydroxylation. C, decay at 500 nm observed upon FIH-catalyzed hydroxylation of 1CA, 2CA, and 3CA substrates.
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Figure 10: Stopped-flow UV-visible absorbance changes upon FIH-catalyzed hydroxylation of HIF-1/2α CAD and ARD substrates. Reaction mixtures containing anaerobic 0.5 mm apo-FIH, 0.4 mm Fe(II), 5 mm 2OG, and 1 mm peptide in HEPES 50 mm (pH 7.5) were rapidly mixed with O2-saturated buffer at 5 °C in 1:1 ratio and analyzed using a photodiode array detector. The data were fitted with a double exponential function. A, decay at 500 nm (representing enzyme·substrate complex depletion) observed upon FIH-catalyzed hydroxylation of ARD substrates compared with HIF-1/2α CAD. B, formation and degradation of 310 nm species observed upon FIH-catalyzed HIF-1/2α CAD and ARD hydroxylation. C, decay at 500 nm observed upon FIH-catalyzed hydroxylation of 1CA, 2CA, and 3CA substrates.
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).

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.Under pre-steady-state conditions, the O2-initiated FIH reaction is significantly faster than that of PHD2.

View Article: PubMed Central - PubMed

Affiliation: From the Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom and.

Show MeSH
Related in: MedlinePlus