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Effect of desferrioxamine and metals on the hydroxylases in the oxygen sensing pathway
Author(s) -
Hirsilä Maija,
Koivunen Peppi,
Xu Leon,
Seeley Todd,
Kivirikko Kari I.,
Myllyharju Johanna
Publication year - 2005
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.04-3399fje
Subject(s) - chemistry , recombinant dna , enzyme , cobalt , biochemistry , catalysis , hydroxylation , in vitro , substrate (aquarium) , stereochemistry , biology , organic chemistry , gene , ecology
ABSTRACT Hypoxia‐inducible transcription factor (HIF) is regulated by two oxygen‐dependent events that are catalyzed by the HIF prolyl 4‐hydroxylases (HIF‐P4Hs) and HIF asparaginyl hydroxylase (FIH). We have purified the three recombinant human HIF‐P4Hs to near homogeneity and characterized their catalytic properties and inhibition and those of FIH. The specific activities of the HIF‐P4Hs were at least 40–50 mol/mol/min, and they and FIH catalyzed an uncoupled decarboxylation of 2‐oxoglutarate in the absence of any peptide substrate. The purified HIF‐P4Hs showed considerable activities even without added Fe 2+ , their apparent K m values for iron being markedly lower than that of FIH. Desferrioxamine and several metals were effective inhibitors of FIH, but surprisingly, ineffective inhibitors of the HIF‐P4Hs in vitro, especially of HIF‐P4H‐2. Desferrioxamine and cobalt were more effective in cultured insect cells synthesizing recombinant HIF‐P4H‐2, but complete inhibition was not achieved and most of the enzyme was inactivated irreversibly. Cobalt also rapidly inactivated HIF‐P4Hs during storage at 4°C. The well‐known stabilization of HIF‐α by cobalt and nickel is thus not due to a simple competitive inhibition of HIF‐P4Hs. The effective inhibition of FIH by these metals and zinc probably leads to full transcriptional activity of HIF‐α even in concentrations that produce no stabilization of HIF‐α.