Effect of desferrioxamine and metals on the hydroxylases in the oxygen sensing pathway.
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.
Hirsilä
Maija
M
Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland.
Koivunen
Peppi
P
Xu
Leon
L
Seeley
Todd
T
Kivirikko
Kari I
KI
Myllyharju
Johanna
J
D000595:Amino Acid Sequence; D002460:Cell Line; D003035:Cobalt / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007501:Iron / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009532:Nickel / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology*; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis; D015032:Zinc / Q000494:pharmacology
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008670:Metals; D042461:Vascular Endothelial Growth Factor A; D012710:Serum Albumin, Bovine; D003035:Cobalt; D009532:Nickel; D007501:Iron; D011393:Procollagen-Proline Dioxygenase; D003676:Deferoxamine; D015032:Zinc; D010100:Oxygen