The length of peptide substrates has a marked effect on hydroxylation by the hypoxia-inducible factor prolyl 4-hydroxylases.
Three hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the HIFs by hydroxylating prolines at two separate sites in the oxygen-dependent degradation domain (ODDD) of their alpha subunits. We compared in vitro hydroxylation by purified recombinant human HIF-P4Hs of 19-20- and 35-residue peptides corresponding to the two sites in HIF-alphas and purified recombinant HIF-1alpha and HIF-2alpha ODDDs of 248 and 215 residues. The increase in the length of peptides representing the C-terminal site from 19 to 20 to 35 residues reduced the K(m) values to 90-800 nm, i.e. to 0.7-11% of those for the shorter peptides, whereas those representing the N-terminal site were 10-470 microm, i.e. 10-135%. The K(m) values of HIF-P4H-1 for the recombinant HIF-alpha ODDDs were 10-20 nm, whereas those of HIF-P4H-2 and -3 were 60-140 nm, identical values being found for the wild-type HIF-1alpha ODDD and its N site mutant. The K(m) values for the C site mutant were about 5-10 times higher but only 0.2-3% of those for the 35-residue N site peptides, and this marked difference suggested that the HIF-P4Hs may become bound first to the C-terminal site of an ODDD and that this binding may enhance subsequent binding to the N-terminal site. The K(m) values of HIF-P4H-2 for oxygen determined with the HIF-1alpha ODDD and both its mutants as substrates were all about 100 microm, being 40% of those reported for the three HIF-P4Hs with a 19-residue peptide. Even this value is high compared with tissue O(2) levels, indicating that HIF-P4Hs are effective oxygen sensors.
Koivunen
Peppi
P
Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, FIN-90014, Oulu, Finland.
Hirsilä
Maija
M
Kivirikko
Kari I
KI
Myllyharju
Johanna
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D051793:Hypoxia-Inducible Factor 1 / Q000737:chemistry*; D007313:Insecta; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D051793:Hypoxia-Inducible Factor 1; D010455:Peptides; D011994:Recombinant Proteins; D011393:Procollagen-Proline Dioxygenase; D010100:Oxygen