Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor Inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents - Archive ouverte HAL Access content directly
Journal Articles Biochemical Journal Year : 2010

Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor Inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents

Emily Flashman
  • Function : Author
Sarah L Davies
  • Function : Author
Kar Kheng Yeoh
  • Function : Author

Abstract

The hypoxia inducible factor (HIF) hydroxylases (prolyl hydroxylases, which in humans are PHD isoforms 1-3, and factor inhibiting HIF, FIH) regulate HIF levels and activity. These enzymes are Fe(II)/2-oxoglutarate dependent oxygenases, many of which are stimulated by ascorbate. We have investigated the ascorbate dependence of PHD2-catalysed hydroxylation of two prolyl hydroxylation sites in human HIF-1α, and of FIH-catalysed hydroxylation of asparaginyl hydroxylation sites in HIF-1α and in a consensus ankyrin repeat domain peptide. The initial rate and extent of hydroxylation was increased in the presence of ascorbate for each of these reactions. When ascorbate was replaced with structural analogues, the results revealed that the ascorbate side chain was not important in its contribution to HIF hydroxylase catalysis, whereas modifications to the ene-diol portion of the molecule negated the ability to promote hydroxylation. We investigated whether alternative reducing agents (glutathione and dithiothreitol) could be used to promote HIF hydroxylase activity, and found partial stimulation of hydroxylation in an apparently enzyme and substrate specific manner. The results raise the possibility of developing reducing agents targeted to specific HIF hydroxylase-catalysed reactions.

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hal-00479275 , version 1 (30-04-2010)

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Emily Flashman, Sarah L Davies, Kar Kheng Yeoh, Christopher J Schofield. Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor Inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents. Biochemical Journal, 2010, 427 (1), pp.135-142. ⟨10.1042/BJ20091609⟩. ⟨hal-00479275⟩

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