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J. Biol. Chem., Vol. 281, Issue 39, 28712-28720, September 29, 2006

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

From the Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, FIN-90014, Oulu, Finland

Received for publication, May 15, 2006 , and in revised form, August 1, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 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-s and purified recombinant HIF-1 and HIF-2 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 µM, i.e. 10–135%. The K_m values of HIF-P4H-1 for the recombinant HIF- 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-1 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-1 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The hypoxia-inducible factors (HIFs)² are master regulators of the transcription of more than 100 hypoxia-regulated genes and play central roles in cellular oxygen homeostasis. HIFs are heterodimers that consist of an oxygen-regulated subunit (HIF-) and a stable subunit (HIF-), and both types of subunits are members of the basic helix-loop-helix Per-Arnt-Sim protein family. The human subunit has three isoforms, HIF-1 to HIF-3 (for reviews, see Refs. 1–3). HIF-1 and HIF-2 are synthesized continuously, and hydroxylation of at least one of two critical proline residues in their oxygen-dependent degradation domain (ODDD), Pro⁴⁰² and Pro⁵⁶⁴ in HIF-1, generates a binding site for the von Hippel-Lindau (VHL) ubiquitin-protein isopeptide ligase complex that targets them for rapid proteasomal degradation under normoxic conditions (4–7). This hydroxylation is catalyzed in humans by three recently identified cytoplasmic and nuclear HIF prolyl 4-hydroxylases (HIF-P4Hs 1–3, also named PHD1–3, HPH3-1, and EGLN2, -1, and -3, respectively) (8–10). These are distinct from the well characterized collagen prolyl 4-hydroxylases (C-P4Hs) that reside within the lumen of the endoplasmic reticulum and likewise have three human isoenzymes (11–13). All P4Hs are 2-oxoglutarate dioxygenases and require Fe²⁺, 2-oxoglutarate, O₂, and ascorbate (2, 3, 11, 14). The lack of oxygen inhibits these hydroxylations so that HIF-s are no longer recognized by the VHL protein and degraded but instead dimerize with HIF-. These dimers then become bound to the HIF-responsive elements in various hypoxia-regulated genes (1–3).

Hydroxylation of a specific asparagine in the C-terminal transactivation domain of an HIF- prevents its interaction with the transcriptional coactivator p300, thus inhibiting its full transcriptional activity (15). The asparaginyl hydroxylase responsible for this modification is identical to a previously identified factor inhibiting HIF (FIH) (16, 17). It is also one of the 2-oxoglutarate dioxygenases (16, 17), but its catalytic properties are distinct from those of the HIF-P4Hs (18).

The proline residues to be hydroxylated in HIF-s are located in -Leu-X-X-Leu-Ala-Pro-sequences. It was therefore initially suggested that the HIF-P4Hs may require this conserved core motif (1–3). Early mutagenesis experiments supported this suggestion (8, 10), but subsequent studies indicated that the two leucines can be replaced by many residues (19, 20), alanine being the only relatively but not absolutely strict requirement in addition to the proline itself (20). The HIF-P4Hs require long substrates, the shortest HIF--like peptide hydroxylated by all three recombinant human isoenzymes having 11 residues (21). All three isoenzymes hydroxylated 19-residue peptides with sequences corresponding to those around the C-terminal hydroxylation site in HIF-s, with K_m values of about 5–15 µM for HIF-1 and HIF-3-like peptides and 10–30 µM for an HIF-2-like peptide (21). A subsequent study indicated that a leucine located 10 residues downstream from the proline influences its hydroxylation by HIF-P4Hs (22), which agrees with the previous finding that deletion of two residues, a glutamine and a leucine, from the C terminus of a 19-residue peptide corresponding to the C-terminal hydroxylation site in HIF-1 increased its K_m values for HIF-P4H-1 and -2 but not for HIF-P4H-3 (21). As the 19-residue peptide used to study hydroxylation of the C-terminal site in HIF-2 ended just before this leucine (21), it is unknown whether its slightly higher K_m value relative to those of the HIF-1 and HIF-3 peptides (above) is simply because of the lack of this residue. A 19-residue peptide corresponding to the N-terminal hydroxylation site in HIF-1 had much higher K_m values for HIF-P4H-1 and -2 than the C-terminal site peptide and was not hydroxylated by HIF-P4H-3 to any significant extent at all, whereas a 19-residue peptide corresponding to the N-terminal site in HIF-2 was hydroxylated by all three isoenzymes, but again with distinctly higher K_m values than the corresponding C-terminal site peptide (21).

As the K_m values determined for the 19-residue HIF--like peptides are relatively high (21), and as residues such as a leucine located 10 residues downstream of the proline to be hydroxylated (22) and also some of the acidic residues present in various parts of the 19-residue peptides (19, 20) have been shown to have distinct effects, we studied here whether the K_m values for 35-residue HIF--like peptides and recombinant HIF-1 and HIF-2 fragments of 248 and 215 residues, respectively, differ from those determined for shorter peptides. The HIF- fragments correspond to the ODDDs and contain both proline hydroxylation sites in the same polypeptide, which made it possible to study whether this situation differs from the presence of the two sites in separate peptides. Hydroxylation of only one of the two sites in the HIF-1 ODDD could be studied using its two mutants, P402A and P564A. A recent study has indicated that Pro⁵⁶⁴ in HIF-1 is hydroxylated before Pro⁴⁰² in cultured cells and that hydroxylation of Pro⁴⁰² is inhibited at higher oxygen tensions than that of Pro⁵⁶⁴ (23). We therefore also determined the K_m values of HIF-P4H-2 for O₂ in the hydroxylation of the wild-type and the two mutant HIF-1 ODDDs. Our data indicate that increasing the peptide length from 19 to 20 to 35 residues has a marked effect on the K_m values for peptides representing the C-terminal hydroxylation site in all three HIF- isoforms, whereas more modest or no effects are seen in the case of peptides representing the N-terminal site. The K_m values determined for the C-terminal hydroxylation site in the recombinant HIF-1 ODDD are in most cases still somewhat lower than those for the corresponding 35-residue peptides, whereas the K_m values for its N-terminal site are markedly lower than those for the 35-residue N site peptides, and this site had a low K_m value even for HIF-P4H-3, which had very high K_m values for the corresponding 20- and 35-residue peptides. The K_m value of HIF-P4H-2 for O₂ determined with the HIF-1 ODDD was lower than that reported with a 19-residue peptide, but was still high, about 100 µM, and no difference was found in this K_m value when determined with the wild-type or the two mutant ODDDs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Expression and Purification of Recombinant HIF-P4Hs—FLAG His-tagged HIF-P4H-1–3 (24) were expressed in H5 insect cells cultured in suspension or on plates in Sf900IISFM serum-free medium (Invitrogen). The cells, seeded at a density of 1 x 10⁶/ml, were infected with recombinant baculoviruses at a multiplicity of 5, harvested 72 h after infection, and then were washed with a solution of 0.15 M NaCl and 0.02 M phosphate, pH 7.4, and homogenized in a buffer containing 0.15 M NaCl, 0.1 M glycine, 10 µM dithiothreitol, 0.1% Triton X-100, 1 or 5 µM FeSO₄, and 0.01 M Tris, pH 7.8, supplemented with Complete EDTA-free protease inhibitor mixture (Roche Applied Science). The recombinant HIF-P4Hs were purified from the soluble fractions with an anti-FLAG M2 affinity gel (Sigma) (24).

Expression and Purification of Recombinant ODDDs—The primer pairs 5'-GCGCATATGCAAACAGAATGTGTCCTTAAACCGG-3' and 5'-GCGCTCGAGCTGGAATACTGTAACTGTGCTTTGAGG-3', and 5'-GCGCATATGCAGACTGAATCCCTGTTCAAGCCCC-3' and 5'-GCGCTCGAGCTGGAAGATGTTTGTCATGGCACTGAAGC-3' (NdeI and XhoI sites underlined) were used to amplify cDNA fragments encoding the ODDDs of HIF-1 and HIF-2, residues Gln³⁵⁶–Gln⁶⁰³ and Gln³⁵⁸–Gln⁵⁷², respectively, by PCR using the plasmids d38e6 and d28e6 containing full-length HIF-1 and HIF-2 cDNAs (a gift from FibroGen Inc.) as templates. The NdeI-XhoI-digested PCR fragments were cloned into a similarly cut pET-22b(+) Escherichia coli expression vector (Novagen) in-frame with a C-terminal histidine tag. Site-directed mutagenesis of the HIF-1 ODDD to P402A and P564A mutant ODDDs and to P402A/P564A double mutant ODDD was performed using the QuikChange^TM kit (Stratagene). The expression plasmids were transformed into the E. coli BL21(DE3) strain (Novagen), and the cells were grown at 37 °C to an absorbance of 0.4 at 600 nm, and expression was induced with 1 mM isopropyl 1-thio--D-galactopyranoside. Cells were harvested after a 3-h induction, solubilized, and boiled in the SDS-PAGE sample buffer for 5 min, and expression of the recombinant ODDDs was analyzed by 12% SDS-PAGE under reducing conditions followed by Coomassie Blue staining.

The recombinant ODDDs were purified from 150–300-ml cultures on a chelating Sepharose column charged with Ni²⁺ (ProBond, Invitrogen). The cells were harvested after a 3-h induction, suspended in 1/20 volume of a 0.5 M NaCl, 0.1% Triton X-100, and 20 mM Tris-HCl buffer, pH 8, supplemented with Complete EDTA-free protease inhibitor mixture (Roche Applied Science), disrupted by sonication, and centrifuged at 17,000 x g for 20 min, and the soluble fractions were applied to a chelating Sepharose column stabilized with a solution of 0.5 M NaCl and 20 mM Tris-HCl, pH 8. The column was washed with the same buffer containing 0.02 M imidazole and eluted with a 150-ml linear imidazole gradient (0.02–0.3 M), and the fractions were analyzed by 12% SDS-PAGE. The fractions containing the recombinant ODDD polypeptides were pooled and concentrated using Amicon ultracentrifugal devices with M_r 5,000 cut-off membrane (Millipore) and applied to a Superdex S-200 column (Amersham Biosciences) in a 0.3 M NaCl, 5 mM dithiothreitol, 20 mM Tris buffer, pH 8.0. Fractions of 2 ml were collected and analyzed by 12% SDS-PAGE. Those containing the pure ODDD polypeptides were pooled, and their buffer was changed to 50 mM Tris-HCl, pH 8, by concentrating as above, and the protein concentrations were measured by RotiQuant (Carl Roth GmbH).

HIF-P4H Activity Assays—HIF-P4H activity was assayed by a method based on measurement of the hydroxylation-coupled stoichiometric release of ¹⁴CO₂ from 2-oxo[1-¹⁴C]glutarate in a final reaction volume of 0.5 ml (21). The K_m values of the purified enzymes for the synthetic HIF-1, HIF-2, and HIF-3 peptides (Table 1), the recombinant ODDD polypeptides, and of HIF-P4H-2 for O₂ with the wild-type HIF-1 ODDD and its P402A and P564A mutants as substrates were determined as described previously (21). The purities of the synthetic HIF- peptides (Innovagen) were about 80%. The K_m values for the 19–20-residue and 35-residue peptides and the ODDD polypeptides were determined using 2-oxoglutarate with a specific activity of 1100 or 11,100, 11,100 or 55,500, and 55,500 dpm/nmol, respectively. The relative V_max values of each HIF-P4H for the various peptide substrates were determined with respect to that obtained with the 19-residue peptide representing the C-terminal hydroxylation site of HIF-1 in the same experiment.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
K_m Values of the Three HIF-P4Hs for 35-Residue Peptides Representing the C-terminal Hydroxylation Site in HIF-1, HIF-2, and HIF-3 Are Markedly Lower than Those for the Corresponding 19–20 Residue Peptides—The human HIF-P4H-1–3 were expressed as FLAG His-tagged recombinant enzymes in insect cells and purified to near homogeneity by anti-FLAG affinity chromatography (24). The 35-residue peptides were designed to contain 17 amino acids on each side of the proline to be hydroxylated (Table 1). The K_m values of the HIF-P4Hs were determined by a method based on measurement of the hydroxylation-coupled stoichiometric release of ¹⁴CO₂ from 2-oxo[1-¹⁴C]glutarate (21, 24). These values were compared with those determined previously for the corresponding 19- or 20-residue peptides (21), and additional values were determined for some of the short peptides. As the 19-residue HIF-2 peptide studied previously ended just before a leucine subsequently reported to influence hydroxylation (22), we also determined the K_m values for a 20-residue peptide containing this leucine (Table 1). Its K_m values were about 50–70% of those for the 19-residue peptide, indicating that the leucine had a distinct although relatively modest effect (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Km and Vmax values of the HIF-P4H isoenzymes for peptides representing the C-terminal hydroxylation site in HIF-1, HIF-2, and HIF-3 The values are means ± S.D.

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Determination of Km values for 35-residue peptides and HIF- ODDDs as substrates of HIF-P4Hs. A, effect of the concentration of a 35-residue peptide representing the C-terminal hydroxylation site in HIF-1 on the reaction velocity of HIF-P4H-3. B, effect of the concentration of a 35-residue peptide representing the N-terminal hydroxylation site in HIF-1 on the reaction velocity of HIF-P4H-2. C, effect of the concentration of the HIF-1 ODDD on the reaction velocity of HIF-P4H-2. D, effect of the concentration of the P564A mutant HIF-1 ODDD on the reaction velocity of HIF-P4H-2. The Km values of the HIF-P4Hs for the substrates were calculated from the double-reciprocal plots (insets). The Vmax values obtained from these plots cannot be compared directly, as the determinations were made in experiments with different enzyme preparations and with 2-oxoglutarate preparations of different specific activities.

View this table: [in this window] [in a new window]   TABLE 3 Km and Vmax values of the HIF-P4H isoenzymes for peptides representing the N-terminal hydroxylation site in HIF-1 and HIF-2 The values are means ± S.D.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Schematic representation of the recombinant ODDDs used here. The recombinant HIF-1 and HIF-2 ODDDs consist of residues Gln356–Gln603 and Gln358–Gln572 of the full-length HIF-1 and HIF-2, respectively. They are fused to a C-terminal histidine tag that is separated from the ODDD sequences by the amino acids (aa) Leu and Glu derived from the 3' XhoI cloning site. The HIF-1 ODDD mutants, P402A, P564A, and P402A/P564A were generated by site-directed mutagenesis. In addition to the ODDDs, the basic helix-loop-helix (bHLH), per-ARNT-Sim (PAS), N and C-terminal transactivation domains (NAC and CAD), and the inhibitory domain (ID) are indicated in the full-length HIF-1 and HIF-2.

The Presence of Both Hydroxylation Sites in a Single Polypeptide Has a Marked Effect on K_m Values for the N-terminal Site—Hydroxylation of the two sites in a single polypeptide was studied by using recombinant HIF-1 and HIF-2 fragments of 248 and 215 residues, respectively, which correspond to their ODDDs (Fig. 2). These recombinant polypeptides were expressed in E. coli in forms containing a C-terminal His tag and purified to homogeneity from the soluble fraction of the cell homogenates on a Ni²⁺-charged chelating Sepharose column followed by gel filtration on Superdex S-200 (Fig. 3). Hydroxylation of only one of the two prolines in the HIF-1 ODDD was studied using its two mutants, P402A and P564A (Fig. 2), which were expressed and purified to homogeneity in the same manner as the wild-type fragment (Fig. 3).

View larger version (34K): [in this window] [in a new window]   FIGURE 3. Expression and purification of the recombinant ODDDs. A, expression of recombinant HIF-1 ODDD (lane 2), HIF-2 ODDD (lane 4), P402A mutant HIF-1 ODDD (lane 6), and P564A mutant HIF-1 ODDD (lane 8) was induced in the E. coli BL21(DE3) strain with isopropyl 1-thio--D-galactopyranoside. Cells were harvested after a 3-h induction, solubilized in the SDS-PAGE sample buffer, and analyzed by 12% SDS-PAGE under reducing conditions followed by Coomassie Blue staining. Control samples before the induction are shown in lanes 1, 3, 5, and 7. B, the recombinant HIF-1 ODDD (lane 1), HIF-2 ODDD (lane 2), P402A mutant HIF-1 ODDD (lane 3), and P564A mutant HIF-1ODDD (lane 4) were purified on a chelating Sepharose column charged with Ni2+ followed by gel filtration, and the purified polypeptides were analyzed by 12% SDS-PAGE under reducing conditions followed by Coomassie Blue staining.

View this table: [in this window] [in a new window]   TABLE 4 Km and Vmax values of the HIF-P4H isoenzymes for recombinant HIF-1 and HIF-2 ODDDs and mutants of the HIF-1 ODDD The values are means ± S.D.

As the difference in the K_m values of HIF-P4H-3 for the P564A and P402A mutant HIF-1 ODDDs was markedly smaller than that in the K_m values for the 35-residue HIF-1 N and C site peptides, we wanted to verify the former difference with a separate assay. The HIF-P4H-3 activity was determined by using L-[2,3,4,5-³H]proline-labeled substrates and measuring the amount of 4-hydroxy[³H]proline formed. Wild-type, P402A and P564A mutant, and P402A/P564A double mutant HIF-1 ODDDs were produced in the presence of L-[2,3,4,5-³H]proline in a rabbit reticulocyte lysate and subjected to hydroxylation by equal amounts of the purified recombinant HIF-P4H-3. Based on the amounts of [³H]proline incorporated and SDS-PAGE analysis followed by fluorography (data not shown), equal amounts of the ODDD and its three mutants were produced in the rabbit reticulocyte lysate, and based on the specific activity of the labeled proline we estimated that the final concentrations of the ODDDs in the activity assay were about 1 nM, i.e. much below their K_m values and thus nonsaturating. HIF-P4H-3 hydroxylated the P402A mutant ODDD as effectively as the wild-type ODDD (Table 5); this finding agreed with our suggestion (above) that hydroxylation of the wild-type ODDD reflected that of its C-terminal site. In contrast, hydroxylation of the P564A mutant ODDD was much less effective, the amount of 4-hydroxy[³H]proline formed being about 8% of those of the P402A mutant and wild-type ODDDs (Table 5). This difference is in a good agreement with the 10-fold difference in the K_m values and 2-fold difference in the V_max values between the P564A mutant ODDD and the P402A mutant and wild-type ODDDs (Table 4).

View this table: [in this window] [in a new window]   TABLE 5 4-Hydroxy[3H]proline formed in wild-type and mutant HIF-1 ODDDs by HIF-P4H-3 The ODDDs were produced in the presence of L-[2,3,4,5-3H]proline in a rabbit reticulocyte lysate and subjected to hydroxylation by purified recombinant HIF-P4H-3. The amount of 4-hydroxy[3H]proline formed was analyzed by a specific radiochemical assay for 4-hydroxyproline. Similar values were obtained from three independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The present data indicate that the length of the peptide substrates has a marked effect on the K_m values of the HIF-P4Hs, which decrease with increasing chain length, whereas the V_max values show only minor changes and, surprisingly, decrease rather than increase when the values for the 19–20-residue peptides are compared with those for the ODDDs. A similar situation has been reported previously for the C-P4Hs in that their K_m values for (X-Pro-Gly)_n peptide substrates and nonhydroxylated collagen fragments decrease markedly with increasing chain length up to at least about 500 residues, whereas the V_max values are not affected (11, 14, 27). Although the (X-Pro-Gly)_n peptides have a hydroxylatable proline at every 3rd position, experiments with nonhydroxylated collagen fragments have demonstrated that their K_m values are related to the total number of their -X-Y-Gly-triplets, i.e. chain length, rather than to the hydroxylatable -X-Pro-Gly-triplets alone (27). As the C-P4Hs require their substrates to be in a nontriple helical conformation (11, 14), the need for long substrates cannot be explained by their enhanced formation of a correct three-dimensional structure, and the reasons for the preferential interaction of the C-P4Hs with long peptides are still not fully understood. Native full-length HIF-1 ODDD has been found to be unstructured in solution under physiological conditions and to belong to the family of native unfolded proteins (28). Hydroxylated HIF-1-like peptides become bound to the VHL protein in an extended conformation, with residues 560–567 and 571–577 forming two distinct binding sites (29, 30). It therefore seems highly likely that HIF--like peptides and full-length HIF- ODDDs also become bound to the HIF-P4Hs in an unfolded conformation, and thus the requirement of long peptide substrates cannot be explained by their effective formation of correct three-dimensional structures.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Determination of the Km value for O2 with a recombinant HIF-1 ODDD as a substrate. A, effect of O2 concentration on the reaction velocity of HIF-P4H-2 with the recombinant HIF-1 ODDD as a substrate. B, effect of O2 concentration on the reaction velocity of HIF-P4H-2 with the recombinant P564A mutant HIF-1ODDD as a substrate. The Km values for O2 were calculated from the double-reciprocal plots (insets).

The effects of increasing the peptide length from 19–20 to 35 residues were distinctly different in the peptides representing the C-terminal and N-terminal hydroxylation sites in HIF-. This increase reduced the K_m values for peptides representing the C-terminal hydroxylation site in almost all cases to only 0.7–5% of those with the shorter peptides, whereas the corresponding changes for peptides representing the N-terminal hydroxylation site were 50–135% with HIF-P4H-2 and -3 and 10–20% with HIF-P4H-1. The K_m values for the 35-residue N site peptides were markedly higher than those for the C site peptides, being 30–125-fold in the cases of HIF-P4H-1 and -2 and 750–5200-fold in the case of HIF-P4H-3, whereas the K_m values for the 20-residue N site peptides were 4–7-fold relative to those for the corresponding C site peptides in the cases of HIF-P4H-1 and -2 and 20–120-fold in the case of HIF-P4H-3. Surprisingly, a further increase in peptide length from 35 to 248 residues caused no further reduction in the K_m value of HIF-P4H-3 for the C-terminal hydroxylation site when studied with the P402A mutant ODDD, whereas decreases to less than 7% and about 20% were seen in the cases of HIF-P4H-1 and -2. In contrast, this increase in peptide length reduced the K_m values of the three HIF-P4Hs for the N-terminal site to 0.2–3%, as studied with the P564A mutant ODDD. Nevertheless, it is not known whether this marked decrease was due entirely to the increase in peptide length or whether it was at least in part due to the presence of both hydroxylation sites in a single polypeptide, even though the C-terminal site was present in the P564A mutant form.

The K_m values of the wild-type and P402A mutant HIF-1 ODDDs were essentially identical, whereas the K_m values for the P564A mutant ODDD were about 5–13 times higher, indicating that the values determined for the wild-type ODDD probably represent those for the C-terminal hydroxylation site and that the HIF-P4Hs interact more effectively with the C-terminal site than with the N-terminal site. Our data thus agree with a recent report indicating that the C-terminal site in HIF-1 is hydroxylated before the N-terminal site in cultured cells (23). Furthermore, the V_max of HIF-P4H-3 for the P564A mutant ODDD was about half that for the wild-type and the P402A mutant ODDD, indicating particularly ineffective hydroxylation of the N-terminal site by HIF-P4H-3. Our hydroxylation experiments with the proline-labeled HIF-1 ODDD and its mutants support these suggestions. It seems highly likely that the HIF-P4Hs may become bound first to the C-terminal site of a full-length ODDD and that this binding may then enhance binding to the N-terminal site. This proposal would be consistent with the marked decrease in K_m values when the length of the N-terminal site peptide was increased from 35 residues to that of the P564A mutant ODDD.

Studies in cultured cells have suggested that the actions of the HIF-P4Hs on different HIF- isoforms are not equivalent, with HIF-P4H-2 having relatively more effect on HIF-1 than on HIF-2 and HIF-P4H-3 having relatively more effect on HIF-2 than on HIF-1 (31). Our data suggest that these differences are not because of differences in the K_m values of the HIF-P4Hs for the corresponding HIF- ODDDs, as HIF-P4H-2 had a slightly higher K_m value for the HIF-1 than for the HIF-2 ODDD, whereas HIF-P4H-3 had identical values for both ODDDs, and these values were between those of HIF-P4H-2 for the two ODDDs. According to our data, HIF-P4H-1 has the lowest K_m values for both the HIF-1 and HIF-2 ODDDs, suggesting that it might interact with them more effectively than the other two isoenzymes. It should be noted, however, that interaction of the HIF-P4Hs with HIF- isoforms in cells is also influenced by the cellular location of the enzymes and their substrates and by the concentrations of the various enzyme proteins.

The K_m values of the HIF-P4Hs for oxygen, when determined with a 19-residue peptide representing the C-terminal hydroxylation site in HIF-1, were very high, 230–250 µM, being slightly above the concentration of dissolved O₂ in the air (21). Although early kinetic studies suggested that the cosubstrates and the peptide substrate may become bound to the C-P4Hs in the order Fe²⁺, 2-oxoglutarate, O₂, and the peptide substrate, the order of binding of the last two reactants was somewhat uncertain (14). A subsequent study indicated that the K_m value of a C-P4H for O₂ is distinctly higher in the uncoupled decarboxylation of 2-oxoglutarate than in the hydroxylation reaction (32), i.e. in C-P4H-catalyzed 2-oxoglutarate decarboxylation without subsequent hydroxylation of the peptide substrate, in which ascorbate acts as an alternative oxygen acceptor (14). This finding suggested that the peptide substrate may in fact become bound to the C-P4Hs in the hydroxylation reaction before O₂. Subsequent mechanistic studies based on the crystal structures of several 2-oxoglutarate dioxygenases have demonstrated that this assumption was correct (2). As HIF-P4H-2 is the most abundant, and therefore most important, HIF-P4H isoenzyme in most cell types (31, 33), we determined here its K_m value for O₂ with the recombinant HIF-1 ODDD as a substrate and obtained a value of about 100 µM, i.e. about 40% of those determined for the three HIF-P4Hs with the 19-residue peptide. In agreement with the order of addition of the cosubstrates to the enzyme, the use of ODDD instead of the 19-residue peptide as a substrate had no effect on the K_m values for Fe²⁺ and 2-oxoglutarate or the IC₅₀ for the 2-oxoglutarate analogue inhibitor pyridine 2,4-dicarboxylate (21, 24), i.e. reactants that become bound to the enzyme before the peptide substrate.

The K_m value of FIH for O₂, when determined with a 35-residue peptide as a substrate, was about 90 µM (18), being about one-third of those determined for the HIF-P4Hs with the 19-residue peptide as a substrate (21). This difference suggested that a minor decrease in the O₂ concentration is likely to influence primarily the activities of the HIF-P4Hs, whereas a larger decrease in the O₂ concentration is needed for a significant decrease in the activity of FIH (18). As FIH appears to require even longer peptide substrates than the HIF-P4Hs (16, 18) and is likely to have a reaction mechanism identical to that of the HIF-P4Hs, it seems likely that the K_m of FIH for O₂ determined with a long HIF- fragment would also be lower than that determined with the 35-residue peptide. It thus seems possible that the difference in the K_m values of the HIF-P4Hs and FIH for O₂ is a biologically relevant difference.

The K_m value of HIF-P4H-2 for O₂ determined here is still very high and is above the oxygen concentrations found in almost all tissues under normoxic conditions in vivo (34). Accordingly, the in vivo activities of the HIF-P4Hs are likely to be limited by O₂ and are likely to be altered by changes in its concentrations in a very sensitive way, making these enzymes effective oxygen sensors. It should be noted, however, that HIF-P4H-2 has considerable activity levels even under O₂ concentrations that are much lower than the K_m value, e.g. at 20 and 10 µM (Fig. 4), which correspond to about 2 and 1% O₂ in the air. This situation explains why the HIF-P4Hs have considerable activity in cultured cells even in the presence of low O₂ concentrations (23). Although hydroxylation of the N-terminal site of HIF-1 has been shown to decrease at limiting O₂ tensions in cultured cells at higher O₂ tensions than that of the C-terminal site (23), we found no difference in the K_m values of HIF-P4H-2 for O₂ when determined with the wild-type, P564A, or P402A mutant HIF-1 ODDD, suggesting that the difference seen in cultured cells may be due to the lower affinity of the N-terminal site for the enzyme, and possibly local changes in O₂ concentrations, rather than differences in the K_m values for the two hydroxylation sites.

	FOOTNOTES

 
^* This work was supported by Grant 202469 from the Health Science Council and Grant 44843 from the Finnish Centre of Excellence Programme 2000–2005 of the Academy of Finland, by the S. Juselius Foundation, and by FibroGen Inc. (South San Francisco). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Collagen Research Unit, Biocenter Oulu and Dept. of Medical Biochemistry and Molecular Biology, P. O. Box 5000, University of Oulu, FIN-90014 Oulu, Finland. Tel.: 358-8-537-5740; Fax: 358-8-537-5811; E-mail: johanna.myllyharju{at}oulu.fi.

² The abbreviations used are: HIF, hypoxia-inducible transcription factor; ODDD, oxygen-dependent degradation domain; VHL, von Hippel-Lindau; HIF-P4H, HIF prolyl 4-hydroxylase; C-P4H, collagen prolyl 4-hydroxylase; FIH, HIF asparaginyl hydroxylase.

	ACKNOWLEDGMENTS

 
We thank T. Aatsinki, R. Juntunen, A. Kokko, A. Myllymäki, and E. Lehtimäki for their excellent technical assistance.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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