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 Pro(564) in HIF-1alpha, whereas a Pro(402) peptide had 20-50-fold K-m values for two isoenzymes but was not hydroxylated by the shortest isoenzyme at all; this difference was not explained by the two prolines being in a -Pro(402)-Ala- and -Pro(564)-Tyr- sequence. All the HIF-P4Hs-hydroxylated peptides corresponding to two of three potential sites in HIF-2alpha and one in HIF-3alpha. The K-m values for O-2 were slightly above its atmospheric concentration, indicating that the HIF-P4Hs are effective oxygen sensors. Small molecule inhibitors of collagen P4Hs also inhibited the HIF-P4Hs, but with distinctly different K-i values, indicating that it should be possible to develop specific inhibitors for each class of P4Hs and possibly even for the individual HIF-P4Hs.