Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay

Abstract

The activity and levels of the metazoan HIF (hypoxia-inducible factor) are regulated by its hydroxylation, catalysed by 2OG (2-oxoglutarate)- and Fe(II)-dependent dioxygenases. An oxygen consumption assay was developed and used to study the relationship between HIF hydroxylase activity and oxygen concentration for recombinant forms of two human HIF hydroxylases, PHD2 (prolyl hydroxylase domain-containing protein 2) and FIH (factor inhibiting HIF), and compared with two other 2OG-dependent dioxygenases. Although there are caveats on the absolute values, the apparent K(m) (oxygen) values for PHD2 and FIH were within the range observed for other 2OG oxygenases. Recombinant protein substrates were found to have lower apparent K(m) (oxygen) values compared with shorter synthetic peptides of HIF. The analyses also suggest that human PHD2 is selective for fragments of the C-terminal over the N-terminal oxygen-dependent degradation domain of HIF-1alpha. The present results, albeit obtained under non-physiological conditions, imply that the apparent K(m) (oxygen) values of the HIF hydroxylases enable them to act as oxygen sensors providing their in vivo capacity is appropriately matched to a hydroxylation-sensitive signalling pathway.

Figure 1. Reactions catalysed by the non-haem Fe(II)- and 2-OG-dependent dioxygenases used in the present study

(A) PHDs, (B) FIH, (C) TauD and (D) mPAHX.

Figure 2. Kinetic studies of the GOX using the oxygen consumption assay at 25 °C in 50 mM NaOAc sodium acetate (pH 5.5)

GOX activity with different concentrations of (A) glucose and (B) oxygen.

Figure 3. Kinetic studies of the HIF hydroxylases using the oxygen consumption assay at 37 °C in 50 mM Tris/HCl (pH 7.5)

His₆–PHD2^181–426 activity was assayed with different concentrations of (A) His₆–HIF-1α^530–652 CODD and (B) HIF-1α^556–574 CODD.

Figure 4. SPR data for PHD2^181–426 binding to His₆–HIF-1α^530–698 CODD and His₆–HIF-1α^344–503 NODD

(A) PHD2 (250 nM, 500 nM, 750 nM and 1 μM) binding to His₆–HIF-1α^530–698 CODD: dissociation rate constant 0.186±0.04 s⁻¹. (B) PHD2 (1, 1.5, 2 and 2.5 μM) binding to His₆–HIF-1α^344–503 NODD: dissociation rate constant 0.697±0.24 s⁻¹.

Figure 5. Kinetic studies of the HIF hydroxylases using the oxygen depletion assay at 37 °C in 50 mM Tris/HCl (pH 7.5)

The oxygen dependencies of (A) His₆–PHD2^181–426 with His₆–HIF-1α^530–698 CODD and (B) His₆–FIH with His₆–HIF-1α^653–826 CAD.