Characterization of the Human Prolyl 4-Hydroxylases That Modify the Hypoxia-inducible Factor

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-alpha 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 HIF-P4Hs expressed in insect cells required peptides of more than 8 residues, distinct differences being found between isoenzymes. All the HIF-P4Hs hydroxylated peptides corresponding to Pro564 in HIF-1alpha, whereas a Pro402 peptide had 20-50-fold Km values for two isoenzymes but was…