The Length of Peptide Substrates Has a Marked Effect on Hydroxylation by the Hypoxia-inducible Factor Prolyl 4-Hydroxylases

• Published in: The Journal of biological chemistry 2006-09, Vol.281 (39), p.28712-28720
• Main Authors: Koivunen, Peppi, Hirsilä, Maija, Kivirikko, Kari I., Myllyharju, Johanna
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Dose-Response Relationship, Drug; Hypoxia-Inducible Factor 1 - chemistry; Insecta; Kinetics; Molecular Sequence Data; Mutation; Oxygen - metabolism; Peptides - chemistry; Procollagen-Proline Dioxygenase - chemistry; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M604628200

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 α 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-αs and purified recombinant HIF-1α and HIF-2α 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 Km 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 μm, i.e. 10–135%. The Km values of HIF-P4H-1 for the recombinant HIF-α 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-1α ODDD and its N site mutant. The Km 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 Km values of HIF-P4H-2 for oxygen determined with the HIF-1α ODDD and both its mutants as substrates were all about 100 μm, being 40% of those reported for the three HIF-P4Hs with a 19-residue peptide. Even this value is high compared with tissue O2 levels, indicating that HIF-P4Hs are effective oxygen sensors.