Many Amino Acid Substitutions in a Hypoxia-inducible Transcription Factor (HIF)-1α-like Peptide Cause Only Minor Changes in Its Hydroxylation by the HIF Prolyl 4-Hydroxylases
2004; Elsevier BV; Volume: 279; Issue: 53 Linguagem: Inglês
10.1074/jbc.m410287200
ISSN1083-351X
AutoresDongxia Li, Maija Hirsilä, Peppi Koivunen, Mitchell C. Brenner, Leon Xu, Charles Yang, Kari I. Kivirikko, Johanna Myllyharju,
Tópico(s)Adipose Tissue and Metabolism
ResumoThree human prolyl 4-hydroxylases (P4Hs) regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylating a Leu-Xaa-Xaa-Leu-Ala-Pro motif. We report here that the two leucines in the Leu-Glu-Met-Leu-Ala-Pro core motif of a 20-residue peptide corresponding to the sequence around Pro564 in HIF-1α can be replaced by many residues with no or only a modest decrease in its substrate properties or in some cases even a slight increase. The glutamate and methionine could be substituted by almost any residue, eight amino acids in the former position and four in the latter being even better for HIF-P4H-3 than the wild-type residues. Alanine was by far the strictest requirement, because no residue could fully substitute for it in the case of HIF-P4H-1, and only serine or isoleucine, valine, and serine did this in the cases of HIF-P4Hs 2 and 3. Peptides with more than one substitution, having the core sequences Trp-Glu-Met-Val-Ala-Pro, Tyr-Glu-Met-Ile-Ala-Pro, Ile-Glu-Met-Ile-Ala-Pro, Trp-Glu-Met-Val-Ser-Pro, and Trp-Glu-Ala-Val-Ser-Pro were in most cases equally as good or almost as good substrates as the wild-type peptide. The acidic residues present in the 20-residue peptide also played a distinct role, but alanine substitution for any six of them, and in some combinations even three of them, had no negative effects. Substitution of the proline by 3,4-dehydroproline or l-azetidine-2-carboxylic acid, but not any other residue, led to a high rate of uncoupled 2-oxoglutarate decarboxylation with no hydroxylation. The data obtained for the three HIF-P4Hs in various experiments were in most cases similar, but in some cases HIF-P4H-3 showed distinctly different properties. Three human prolyl 4-hydroxylases (P4Hs) regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylating a Leu-Xaa-Xaa-Leu-Ala-Pro motif. We report here that the two leucines in the Leu-Glu-Met-Leu-Ala-Pro core motif of a 20-residue peptide corresponding to the sequence around Pro564 in HIF-1α can be replaced by many residues with no or only a modest decrease in its substrate properties or in some cases even a slight increase. The glutamate and methionine could be substituted by almost any residue, eight amino acids in the former position and four in the latter being even better for HIF-P4H-3 than the wild-type residues. Alanine was by far the strictest requirement, because no residue could fully substitute for it in the case of HIF-P4H-1, and only serine or isoleucine, valine, and serine did this in the cases of HIF-P4Hs 2 and 3. Peptides with more than one substitution, having the core sequences Trp-Glu-Met-Val-Ala-Pro, Tyr-Glu-Met-Ile-Ala-Pro, Ile-Glu-Met-Ile-Ala-Pro, Trp-Glu-Met-Val-Ser-Pro, and Trp-Glu-Ala-Val-Ser-Pro were in most cases equally as good or almost as good substrates as the wild-type peptide. The acidic residues present in the 20-residue peptide also played a distinct role, but alanine substitution for any six of them, and in some combinations even three of them, had no negative effects. Substitution of the proline by 3,4-dehydroproline or l-azetidine-2-carboxylic acid, but not any other residue, led to a high rate of uncoupled 2-oxoglutarate decarboxylation with no hydroxylation. The data obtained for the three HIF-P4Hs in various experiments were in most cases similar, but in some cases HIF-P4H-3 showed distinctly different properties. The hypoxia-inducible transcription factors (HIFs), 1The abbreviations used are: HIF, hypoxia-inducible transcription factor; HIF-P4H, prolyl 4-hydroxylase hydroxylating HIFα; C-P4H, collagen prolyl 4-hydroxylase; VHL, von Hippel-Lindau; E3, ubiquitin-protein isopeptide ligase. which are essential for the regulation of cellular and systemic oxygen homeostasis, are αβ heterodimers in which both types of subunit are basic helix-loop-helix Per-Arnt-Sim proteins. The human α subunit has three isoforms, HIF-1α to HIF-3α, of which HIF-1α and HIF-2α are expressed constitutively but are rapidly degraded under normoxic conditions (for reviews see Refs. 1Semenza G.L. Cell. 2001; 107: 1-3Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar, 2Kim W. Kaelin Jr., W.G. Curr. Opin. Genet. Dev. 2003; 13: 55-60Crossref PubMed Scopus (166) Google Scholar, 3Poellinger L. Johnson R.S. Curr. Opin. Genet. Dev. 2004; 14: 81-85Crossref PubMed Scopus (138) Google Scholar, 4Schofield C.J. Ratcliffe P.J. Nat. Rev. Mol. Cell. Biol. 2004; 5: 343-354Crossref PubMed Scopus (1637) Google Scholar). This degradation is mediated by the oxygen-sensitive degradation domain, which contains two critical proline residues. Hydroxylation of at least one of these to 4-hydroxyproline is essential for the binding of HIFα to the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex and for the subsequent rapid proteasomal degradation (5Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3917) Google Scholar, 6Jaakkola P. Mole D.R. Tian Y-M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A.V. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4489) Google Scholar, 7Yu F. White S. Zhao Q. Lee F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9630-9635Crossref PubMed Scopus (652) Google Scholar, 8Masson N. William C. Maxwell P.H. Pugh C.W. Ratcliffe P.J. EMBO J. 2001; 20: 5197-5206Crossref PubMed Scopus (862) Google Scholar). This hydroxylation is catalyzed in humans by three recently identified cytoplasmic and nuclear HIF prolyl 4-hydroxylases (HIF-P4Hs) (9Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2130) Google Scholar, 10Epstein A.C.R. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y-M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar, 11Ivan M. Haberberger T. Gervasi D.C. Michelson K.S. Günzler V. Kondo K. Yang H. Sorokina I. Conaway R.C. Conaway J.W. Kaelin Jr., W.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13459-13464Crossref PubMed Scopus (492) Google Scholar), which are distinct from the well characterized endoplasmic reticulum lumenal collagen prolyl 4-hydroxylases (C-P4Hs) (12Kivirikko K.I. Myllyharju J. Matrix Biol. 1998; 16: 357-368Crossref PubMed Scopus (235) Google Scholar, 13Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Related Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar, 14Myllyharju J. Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (338) Google Scholar, 15Kukkola L. Hieta R. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 47685-47693Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 16Van Den Diepstraten C. Papay K. Bolender Z. Brown A. Pickering G. Circulation. 2003; 108: 508-511Crossref PubMed Scopus (44) Google Scholar). All P4Hs belong to the family of 2-oxoglutarate dioxygenases and require Fe2+, 2-oxoglutarate, O2, and ascorbate (12Kivirikko K.I. Myllyharju J. Matrix Biol. 1998; 16: 357-368Crossref PubMed Scopus (235) Google Scholar, 13Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Related Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar, 14Myllyharju J. Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (338) Google Scholar). The Km values of the three human HIF-P4Hs for O2 are slightly above the concentration of dissolved O2 in the air (17Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar), and thus even a small decrease in the O2 concentration will inhibit their activities, leading to stabilization of HIFα and dimer formation with HIFβ. The dimer is then translocated to the nucleus, where it becomes bound to the HIF-responsive elements in a number of hypoxia-inducible genes, such as those for erythropoietin, vascular endothelial growth factor, glycolytic enzymes, and glucose transporters (1Semenza G.L. Cell. 2001; 107: 1-3Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar, 2Kim W. Kaelin Jr., W.G. Curr. Opin. Genet. Dev. 2003; 13: 55-60Crossref PubMed Scopus (166) Google Scholar, 3Poellinger L. Johnson R.S. Curr. Opin. Genet. Dev. 2004; 14: 81-85Crossref PubMed Scopus (138) Google Scholar, 4Schofield C.J. Ratcliffe P.J. Nat. Rev. Mol. Cell. Biol. 2004; 5: 343-354Crossref PubMed Scopus (1637) Google Scholar). The two critical proline residues in human HIF-1α, Pro402 and Pro564, are located in the Leu-Thr-Leu-Leu-Ala-Pro-Ala and Leu-Glu-Met-Leu-Ala-Pro-Tyr sequences, respectively. Pro564 is the principal hydroxylation site (8Masson N. William C. Maxwell P.H. Pugh C.W. Ratcliffe P.J. EMBO J. 2001; 20: 5197-5206Crossref PubMed Scopus (862) Google Scholar, 10Epstein A.C.R. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y-M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar), the Km values of HIF-P4Hs 1 and 2 for a 19-residue peptide corresponding to the N-terminal hydroxylation site being ∼20–50 times higher than those for a peptide corresponding to the C-terminal site, whereas HIF-P4H-3 did not hydroxylate the N-terminal peptide at all (17Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). All three HIF-P4Hs also hydroxylated 19-residue peptides corresponding to the Cand N-terminal hydroxylation sites in HIF-2α and one in HIF-3α, with the sequences Leu-Glu-Thr-Leu-Ala-Pro-Tyr, Leu-Ala-Gln-Leu-Ala-Pro-Thr, and Leu-Glu-Met-Leu-Ala-Pro-Tyr, respectively, although the second of these had distinctly higher Km values than the other two (17Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). These data are in agreement with the suggestion that the hydroxylation may involve a conserved core sequence Leu-Xaa-Xaa-Leu-Ala-Pro (8Masson N. William C. Maxwell P.H. Pugh C.W. Ratcliffe P.J. EMBO J. 2001; 20: 5197-5206Crossref PubMed Scopus (862) Google Scholar, 10Epstein A.C.R. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y-M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar). Initial mutagenesis experiments in fact indicated that substitution of Leu562 by Ala or Ala563 by Gly may prevent any hydroxylation (5Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3917) Google Scholar, 9Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2130) Google Scholar). A more recent study has demonstrated, however, that Leu562 → Val, Leu562 → Ala, and Ala563 → Ser mutants were utilized by the three HIF-P4Hs only marginally less effectively than a wild-type substrate, whereas Leu559 → Val was utilized somewhat less effectively (18Huang J. Zhao Q. Mooney S.M. Lee F.S. J. Biol. Chem. 2002; 277: 39792-39800Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Leu574 has recently been identified as an additional important residue for hydroxylation (19Kageyama Y. Koshiji M. To K.K.W. Tian Y-M. Ratcliffe P.J. Huang L.E. Faseb J. 2004; 18: 1028-1030Crossref PubMed Scopus (62) Google Scholar), which agrees with the previous finding that the deletion of two residues, Gln573 and Leu574, from the C terminus of a 19-residue HIF-1α C-terminal peptide increased the Km values for HIF-P4Hs 1 and 2 ∼9- and 7-fold, respectively, although the Km for HIF-P4H-3 was unaltered, and the Vmax values for all three isoenzymes were likewise unaffected (17Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). Pro402 and Pro564 have several acidic residues in their vicinity, but mutation of Asp556, Asp558, Glu560, Asp569, or Asp570 to asparagine appeared to have little effect on the hydroxylation (6Jaakkola P. Mole D.R. Tian Y-M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A.V. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4489) Google Scholar). An Asp558 → Ala and Glu560 → Gln double mutant also served as a substrate, although less efficiently than the wild-type substrate, whereas an Asp569 → Asn, Asp570 → Asn, and Asp571 → Asn triple mutant failed to act as a substrate at all (18Huang J. Zhao Q. Mooney S.M. Lee F.S. J. Biol. Chem. 2002; 277: 39792-39800Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). The C-P4Hs catalyze an uncoupled decarboxylation of 2-oxoglutarate in the presence of all cosubstrates but in the absence of any peptide substrate at a rate that is ∼0.5–1% of that of the hydroxylation reaction observed in the presence of a saturating concentration of the peptide substrate (12Kivirikko K.I. Myllyharju J. Matrix Biol. 1998; 16: 357-368Crossref PubMed Scopus (235) Google Scholar, 13Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Related Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar, 14Myllyharju J. Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (338) Google Scholar, 20Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (164) Google Scholar). Some peptides that become bound to the C-P4Hs but do not act as substrates enhance the rate of the uncoupled decarboxylation, but it still remains below ∼2% of that of the complete reaction (12Kivirikko K.I. Myllyharju J. Matrix Biol. 1998; 16: 357-368Crossref PubMed Scopus (235) Google Scholar, 13Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Related Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar, 14Myllyharju J. Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (338) Google Scholar). However, substitution of the hydroxylatable proline in a peptide substrate by 3,4-dehydroproline gave an uncoupled decarboxylation rate similar to the hydroxylation rate with the nonmodified peptide (21Wu M. Moon H-S. Pirskanen A. Myllyharju J. Kivirikko K.I. Begley T.P. Bioorg. Med. Chem. Lett. 2000; 10: 1511-1514Crossref PubMed Scopus (10) Google Scholar). No data are currently available to indicate whether substitution of the hydroxylatable proline in HIFα model peptides would lead to a similar high rate of uncoupled decarboxylation. We studied here the sequence requirements of the three human HIF-P4Hs by systematically varying each residue preceding the proline in the Leu-Glu-Met-Leu-Ala-Pro sequence of a 20-residue peptide corresponding to the C-terminal hydroxylation site in HIF-1α. In addition, we studied in detail the effects of substitution of any of six of the seven acidic residues in this peptide by alanine, either alone or in combination with other acidic residues and substitution of the hydroxylatable proline by proline analogues. Our data indicate that the two leucines can be replaced by many residues with either no significant decrease or only a modest decrease in substrate properties and that the glutamate and methionine can well be replaced by most, although not all, amino acids, whereas the alanine is a relatively strict requirement. Any of the six acidic residues studied could be substituted by alanine with no decrease in the substrate properties, and even three of them could be substituted in some combinations with no negative effects. Peptides in which the proline was replaced by either 3,4-dehydroproline or l-azetidine-2-carboxylic acid gave a very high rate of uncoupled 2-oxoglutarate decarboxylation. The data obtained with the three HIF-P4Hs were in most cases similar, but in some cases HIF-P4H-3 showed distinctly different properties. Generation of Recombinant Baculoviruses for the Expression of FLAGHis-tagged HIF-P4H Isoenzymes—The FLAGHis tag was amplified by PCR from the plasmid d28e6 (Fibrogen Inc.) and cloned into the 3′ ends before the stop codons of the recombinant human HIF-P4H isoenzymes 1, 2, and 3 in the pVL baculovirus expression vector (17Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). The recombinant baculovirus expression vectors were cotransfected into Spodoptera frugiperda Sf9 cells with BaculoGold DNA (Pharmingen) by calcium phosphate transfection, and the recombinant baculoviruses were amplified (22Crossen R. Gruenwald S. Baculovirus Expression Vector System: Instruction Manual. PharMingen, San Diego, CA1998Google Scholar). Expression of Recombinant HIF-P4H Isoenzymes in Insect Cells and Purification of the Enzymes—Nontagged HIF-P4Hs 1 and 2 (17Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar), glutathione S-transferase-tagged HIF-P4H-3 (17Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar), or FLAGHis-tagged HIF-P4Hs 1–3 were expressed in Sf9 or H5 cells cultured in suspension in Sf900IISFM serum-free medium (Invitrogen). The cells, seeded at a density of 1 × 106/ml, were infected with the corresponding viruses at a multiplicity of 5, harvested 72 h after infection, washed with a solution of 0.15 m NaCl and 0.02 m phosphate, pH 7.4, and homogenized in a 0.1 m NaCl, 0.1 m glycine, 10 μm dithiothreitol, 0.1% Triton X-100, and 0.01 m Tris buffer, pH 7.8. The soluble fractions were used directly for enzyme activity assays or subjected to further purification. Nontagged HIF-P4H-2 was partially purified by SP Sepharose chromatography. The cells were homogenized in 120 mm NaCl, 20 mm HEPES buffer, pH 7.5, followed by an addition of 0.25% Triton X-100 and 2.5 mm dithiothreitol. The lysate was incubated at 4 °C for 1 h, centrifuged at 20,000 × g for 20 min, and diluted 1:4 in 20 mm HEPES buffer, pH 6.5. The sample was loaded into an SP Fast Flow column (Amersham Biosciences) equilibrated with 20 mm HEPES buffer, pH 6.5. The column was washed with 50 mm NaCl, 20 mm HEPES buffer, pH 6.5, and eluted with a linear gradient of NaCl from 50 to 333 mm in HEPES buffer, pH 6.5, followed by elution with 500 mm NaCl, HEPES buffer, pH 6.5. HIF-P4H-2 was eluted at a 200 mm NaCl concentration. Recombinant HIF-P4H isoenzymes with C-terminal FLAGHis tags were purified to homogeneity with an anti-FLAG M2 affinity gel (Sigma) (23Koivunen P. Hirsilä M. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2004; 279: 9899-9904Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). 2M. Hirsilä, P. Koivunen, L. Xu, T. Seeley, K. I. Kivirikko, and J. Myllyharju, manuscript in preparation. P4H Activity Assays—HIF-P4H activity was assayed by a method based on the hydroxylation-coupled decarboxylation of 2-oxoglutarate (17Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 24Kivirikko K.I. Myllylä R. Methods Enzymol. 1982; 82: 245-304Crossref PubMed Scopus (325) Google Scholar). The synthetic HIF-1α peptide 554DTDLDLEMLAPYIPMDDDFQ573 used as a substrate, and its substituted variants were obtained from SynPep and Mimotopes. The nonpurified peptides used in the initial experiments were obtained from Mimotopes and were used at a concentration of 200 μm, whereas those used in the measurements of Km and Vmax values had a purity of more than 85%. Km and Vmax values for the various HIF peptide substrates were determined at saturating concentrations of the other components, except that the O2 concentration was that of air and thus nonsaturating (17Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). Liquid Chromatography/Mass Spectrometry Analysis of Peptide Hydroxylation—The enzyme activity assays used in this set of experiments were conducted with partially purified HIF-P4H-2, following the standard protocol (17Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 24Kivirikko K.I. Myllylä R. Methods Enzymol. 1982; 82: 245-304Crossref PubMed Scopus (325) Google Scholar), except that the concentration of 2-oxoglutarate was 100 μm, and that of the HIF-1α peptide 554DTDLDLEALAPYIPADDDFQ573 or peptides in which the hydroxylatable Pro564 was substituted by 3,4-dehydroproline or l-azetidine-2-carboxylic acid was likewise 100 μm. The two methionines were substituted by alanines in all of these peptides to eliminate the possible formation of auto-oxidation products. The reaction was carried out at 37 °C for 4 h and stopped by the addition of 1 mm EDTA. Hydroxylation of the peptides was analyzed by liquid chromatography with mass spectrometric detection using a Finnigan LCQ™ DUO LC/MS instrument (Thermo Electron) with electrospray ionization in negative mode. Chromatography was carried out using a YMC Pro Pack C18 (150 × 2.0 mm, 3 μ, 120 Å) column with a gradient of 1–75% acetonitrile in 0.1% formic acid at a flow rate of 0.2 ml/min. Leu559 and Leu562 Can Be Substituted by Many Other Residues with Relatively Small Effects—The 20-residue control peptide used as a substrate in the present study corresponds to the hydroxylation site around Pro564 in HIF-1α and has the sequence 554DTDLDLEMLAPYIPMDDDFQ573. It differs from the 19-residue peptide used previously (17Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar) in that it contains two additional residues in its N terminus and lacks one, Leu574, in its C terminus. The Km values of a purified version of this peptide for HIF-P4Hs 1, 2, and 3 are 15, 25, and 5 μm, respectively (see below). Because of the high cost of synthetic peptides, all of the initial experiments were performed using crude peptide preparations of less than 50% purity. The nominal peptide concentrations used in the initial experiments were 200 μm, but because of the low degree of purity, the true concentrations were less than 100 μm. Thus the concentration of the control peptide used for HIF-P4Hs 1 and 2 may have been less than 4–6.5 times the Km, and that for HIF-P4H-3 may have been less than 20 times the Km. Consequently, the assays may have been insensitive for substitutions that cause only relatively small Km effects with no changes in Vmax, especially in the case of HIF-P4H-3. Enzyme activity was assayed based on measurement of the radioactivity of the 14CO2 formed during the hydroxylation-coupled stoichiometric decarboxylation of 2-oxo[1-14C]glutarate using Triton X-100 buffer extracts of homogenates of insect cells expressing recombinant HIF-P4Hs as sources of the enzymes (17Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar), or in the case of HIF-P4H-2, preparations that had been partially purified from such extracts by one chromatography step. Because the purity of the peptides was low, the reaction rates obtained in the initial experiments are given in Tables I, II, and IV only as >120, 80–120, 50–80, 30–50, 10-30, and 120%TrpIle, Phe80-120%Tyr, Leu, MetTrp, Tyr, Leu, Met, Ala, ValLeu, Val, Ile, Phe, ArgLeu, Val, Ile, Phe, Tyr, Met50-80%Ile, Phe, Ala, Glu, AspGlu, Asp, Cys, Pro, Gln, Ser, ThrTyr, Thr, Lys, MetArg, Thr, Cys30-50%Cys, Pro, Gly, Val, Gln, SerGly, Asn, His, ArgAsn, Cys, Trp, His, Ala, SerLys, Asn, Trp, His, Ala, Ser, Gln10-30%Asn, Thr, His, Arg, LysLysGln, Glu, GlyGlu, Gly 120%Gly, Ser, Val, Ala, Gln, Asn, Arg, LysTyr, Trp, Leu, Val80-120%Most amino acidsOther amino acidsMost amino acidsMost amino acidsAla, SerIle, Ala, Val, Ser50-80%Trp, Val, Phe, Tyr, Cys, Arg, HisProAsp, Glu, ProThr, LysArg, Lys, His, Cys, Gln, Thr30-50%Lys, ProVal, GlnGlu, Met, Phe, Gly, Leu10-30%Met, Gly, Arg, IleTrp, Asp 80%Asp556Asp556 + Asp558Asp556 + Asp558 + Asp570Asp558Asp556 + Glu560Asp556 + Asp558 + Asp571Glu560Asp558 + Glu560Asp556 + Glu560 + Asp570Asp569Asp570 + Asp571Asp556 + Glu560 + Asp571Asp570Asp556 + Asp570Asp558 + Glu560 + Asp570Asp571Asp556 + Asp571Asp558 + Glu560 + Asp571Asp558 + Asp570Asp558 + Asp571Glu560 + Asp570Glu560 + Asp57150-80%Asp556 + Asp569Asp556 + Asp558 + Asp569Asp558 + Asp569Asp556 + Glu560 + Asp569Glu560 + Asp569Asp556 + Asp569 + Asp571Asp569 + Asp570Asp558 + Asp569 + Asp571Asp569 + Asp571Glu560 + Asp569 + Asp571Asp556 + Asp570 + Asp571Asp558 + Asp570 + Asp571Glu560 + Asp570 + Asp57130-50%Asp556 + Asp558 + Glu560Asp556 + Asp558 + Asp569 + Asp571Asp558 + Glu560 + Asp569Asp556 + Asp558 + Asp570 + Asp571Asp556 + Asp569 + Asp570Asp558 + Asp569 + Asp570Glu560 + Asp569 + Asp57010-30%Asp556 + Asp558 + Asp569 + Asp570Asp556 + Glu560 + Asp569 + Asp570Asp556 + Glu560 + Asp569 + Asp571Asp556 + Glu560 + Asp570 + Asp571Asp558 + Glu560 + Asp569 + Asp570Asp558 + Glu560 + Asp569 + Asp571Asp558 + Glu560 + Asp570 + Asp571<10%Asp569 + Asp570 + Asp571Asp556 + Asp569 + Asp570 + Asp571Asp558 + Asp569 + Asp570 + Asp571Glu560 + Asp569 + Asp570 + Asp571a The reaction rates are expressed relative to that determined for the nonmodified control peptide 554DTDLDLEMLAPYIPMDDDFQ573. Open table in a new tab Substitution of Leu559 by other residues gave identical data for HIF-P4Hs 1 and 2 (Table I). Its replacement by tryptophan under the conditions used gave a reaction rate for both isoenzymes that was significantly higher than that obtained with leucine (120–130%), whereas the rates obtained with tyrosine and methionine were similar to that with leucine. Five additional amino acids, isoleucine, phenylalanine, alanine, glutamate and aspartate, gave rates that were 50–80% of that with leucine, whereas the lowest rates, ∼10–30%, were obtained with asparagine, threonine, histidine, arginine, and lysine, thus including all of the basic amino acids (Table I). In the case of HIF-P4H-3, there were two residues, isoleucine and phenylalanine, that gave significantly higher rates than leucine (∼130%), whereas five additional residues, tryptophan, tyrosine, methionine, alanine, and valine, gave rates similar to that with leucine, seven other residues ga
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