The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. Hydroxylation of one or two critical prolines by specific hydroxylases (P4Hs) targets their HIF-␣ subunits for proteasomal degradation. By studying the three human HIF-P4Hs, we found that the longest and shortest isoenzymes have major transcripts encoding inactive polypeptides, which suggest novel regulation by alternative splicing. Recombinant HIFP4Hs expressed in insect cells required peptides of more than 8 residues, distinct differences being found between isoenzymes.

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The activity of hypoxia-inducible transcription factor HIF, an ␣␤ heterodimer that has an essential role in adaptation to low oxygen availability, is regulated by two oxygen-dependent hydroxylation events. Hydroxylation of specific proline residues by HIF prolyl 4-hydroxylases targets the HIF-␣ subunit for proteasomal destruction, whereas hydroxylation of an asparagine in the C-terminal transactivation domain prevents its interaction with the transcriptional coactivator p300. The HIF asparaginyl hydroxylase is identical to a previously known factor inhibiting HIF (FIH). We report here that recombinant FIH has unique catalytic and inhibitory properties when compared with those of the HIF prolyl 4-hydroxylases. FIH was found to require particularly long peptide substrates so that omission of only a few residues from the N or C terminus of a 35-residue HIF-1␣ sequence markedly reduced its substrate activity. Hydroxylation of two HIF-2␣ peptides was far less efficient than that of the corresponding HIF-1␣ peptides. The K m of FIH for O 2 was about 40% of its atmospheric concentration, being about one-third of those of the HIF prolyl 4-hydroxylases but 2.5 times that of the type I collagen prolyl 4-hydroxylase. Several 2-oxoglutarate analogs were found to inhibit FIH but with distinctly different potencies from the HIF prolyl 4-hydroxylases. For example, the two most potent HIF prolyl 4-hydroxylase inhibitors among the compounds studied were the least effective ones for FIH. It should therefore be possible to develop specific small molecule inhibitors for the two enzyme classes involved in the hypoxia response.The hypoxia-inducible transcription factor HIF 1 has a major role in the conserved oxygen-sensitive response pathway that is activated in hypoxic cells. HIF-regulated hypoxia-inducible genes are involved in angiogenesis, vascularization, and anaerobic energy production, for instance. HIFs are ␣␤ heterodimers in which the stability of the ␣ subunit is regulated in an oxygen-dependent manner (for reviews, see Refs. 1-4). Hydroxylation of at least one of two proline residues, Pro 402 and Pro 564 , in the oxygen-dependent degradation domain of human HIF-1␣ mediates the interaction of HIF-␣ with the von Hippel Lindau E3 (ubiquitin-protein isopeptide ligase) ubiquitin ligase complex that targets it for rapid proteasomal degradation under normoxic conditions (5-8). This hydroxylation is catalyzed in humans by three novel cytoplasmic and nuclear HIF prolyl 4-hydroxylases (9 -11) that are distinct from the well characterized collagen prolyl 4-hydroxylases, which reside in the lumen of the endoplasmic reticulum (12-16). Under hypoxic conditions, the oxygen-requiring process of hydroxylation is prevented, and HIF-␣ escapes degradation and dimerizes with HIF-␤, the dimer then recognizing a specific element in the promoters of hypoxia-responsive target genes (1-4).Transcriptional activation in an oxygen-dependent manner is another key step that regulates HIF-␣ activity. Hydroxylation of a specific asparagine residue, Asn 80...

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 Fe2+, their apparent Km 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 degrees C. The well-known stabilization of HIF-alpha 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-alpha even in concentrations that produce no stabilization of HIF-alpha.

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