Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen.
The response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor. Human hypoxia-inducible factor is regulated by four Fe(II)- and 2-oxoglutarate-dependent oxygenases: prolyl hydroxylase domain enzymes 1-3 catalyse hydroxylation of two prolyl-residues in hypoxia-inducible...
| Main Authors: | , , , , , |
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| Format: | Journal article |
| Language: | English |
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2010
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| _version_ | 1826301952660602880 |
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| author | Flashman, E Hoffart, L Hamed, R Bollinger, J Krebs, C Schofield, C |
| author_facet | Flashman, E Hoffart, L Hamed, R Bollinger, J Krebs, C Schofield, C |
| author_sort | Flashman, E |
| collection | OXFORD |
| description | The response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor. Human hypoxia-inducible factor is regulated by four Fe(II)- and 2-oxoglutarate-dependent oxygenases: prolyl hydroxylase domain enzymes 1-3 catalyse hydroxylation of two prolyl-residues in hypoxia-inducible factor, triggering its degradation by the proteasome. Factor inhibiting hypoxia-inducible factor catalyses the hydroxylation of an asparagine-residue in hypoxia-inducible factor, inhibiting its transcriptional activity. Collectively, the hypoxia-inducible factor hydroxylases negatively regulate hypoxia-inducible factor in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 is the most important oxygen sensor in human cells; however, the underlying kinetic basis of the oxygen-sensing function of prolyl hydroxylase domain 2 is unclear. We report analyses of the reaction of prolyl hydroxylase domain 2 with oxygen. Chemical quench/MS experiments demonstrate that reaction of a complex of prolyl hydroxylase domain 2, Fe(II), 2-oxoglutarate and the C-terminal oxygen-dependent degradation domain of hypoxia-inducible factor-α with oxygen to form hydroxylated C-terminal oxygen-dependent degradation domain and succinate is much slower (approximately 100-fold) than for other similarly studied 2-oxoglutarate oxygenases. Stopped flow/UV-visible spectroscopy experiments demonstrate that the reaction produces a relatively stable species absorbing at 320 nm; Mössbauer spectroscopic experiments indicate that this species is likely not a Fe(IV)=O intermediate, as observed for other 2-oxoglutarate oxygenases. Overall, the results obtained suggest that, at least compared to other studied 2-oxoglutarate oxygenases, prolyl hydroxylase domain 2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor. |
| first_indexed | 2024-03-07T05:40:10Z |
| format | Journal article |
| id | oxford-uuid:e54d80f9-45d3-4eeb-b577-db1c815c5fde |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T05:40:10Z |
| publishDate | 2010 |
| record_format | dspace |
| spelling | oxford-uuid:e54d80f9-45d3-4eeb-b577-db1c815c5fde2022-03-27T10:22:58ZEvidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:e54d80f9-45d3-4eeb-b577-db1c815c5fdeEnglishSymplectic Elements at Oxford2010Flashman, EHoffart, LHamed, RBollinger, JKrebs, CSchofield, CThe response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor. Human hypoxia-inducible factor is regulated by four Fe(II)- and 2-oxoglutarate-dependent oxygenases: prolyl hydroxylase domain enzymes 1-3 catalyse hydroxylation of two prolyl-residues in hypoxia-inducible factor, triggering its degradation by the proteasome. Factor inhibiting hypoxia-inducible factor catalyses the hydroxylation of an asparagine-residue in hypoxia-inducible factor, inhibiting its transcriptional activity. Collectively, the hypoxia-inducible factor hydroxylases negatively regulate hypoxia-inducible factor in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 is the most important oxygen sensor in human cells; however, the underlying kinetic basis of the oxygen-sensing function of prolyl hydroxylase domain 2 is unclear. We report analyses of the reaction of prolyl hydroxylase domain 2 with oxygen. Chemical quench/MS experiments demonstrate that reaction of a complex of prolyl hydroxylase domain 2, Fe(II), 2-oxoglutarate and the C-terminal oxygen-dependent degradation domain of hypoxia-inducible factor-α with oxygen to form hydroxylated C-terminal oxygen-dependent degradation domain and succinate is much slower (approximately 100-fold) than for other similarly studied 2-oxoglutarate oxygenases. Stopped flow/UV-visible spectroscopy experiments demonstrate that the reaction produces a relatively stable species absorbing at 320 nm; Mössbauer spectroscopic experiments indicate that this species is likely not a Fe(IV)=O intermediate, as observed for other 2-oxoglutarate oxygenases. Overall, the results obtained suggest that, at least compared to other studied 2-oxoglutarate oxygenases, prolyl hydroxylase domain 2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor. |
| spellingShingle | Flashman, E Hoffart, L Hamed, R Bollinger, J Krebs, C Schofield, C Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen. |
| title | Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen. |
| title_full | Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen. |
| title_fullStr | Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen. |
| title_full_unstemmed | Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen. |
| title_short | Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen. |
| title_sort | evidence for the slow reaction of hypoxia inducible factor prolyl hydroxylase 2 with oxygen |
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