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. 1997 Mar 17;16(6):1173–1180. doi: 10.1093/emboj/16.6.1173

Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase.

J Myllyharju 1, K I Kivirikko 1
PMCID: PMC1169716  PMID: 9135134

Abstract

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha2beta2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens. We converted 16 residues in the human alpha subunit individually to other amino acids, and expressed the mutant polypeptides together with the wild-type beta subunit in insect cells. Asp414Ala and Asp414Asn inactivated the enzyme completely, whereas Asp414Glu increased the K(m) for Fe2+ 15-fold and that for 2-oxoglutarate 5-fold. His412Glu, His483Glu and His483Arg inactivated the tetramer completely, as did Lys493Ala and Lys493His, whereas Lys493Arg increased the K(m) for 2-oxoglutarate 15-fold. His501Arg, His501Lys, His501Asn and His501Gln reduced the enzyme activity by 85-95%; all these mutations increased the K(m) for 2-oxoglutarate 2- to 3-fold and enhanced the rate of uncoupled decarboxylation of 2-oxoglutarate as a percentage of the rate of the complete reaction up to 12-fold. These and other data indicate that His412, Asp414 and His483 provide the three ligands required for the binding of Fe2+ to a catalytic site, while Lys493 provides the residue required for binding of the C-5 carboxyl group of 2-oxoglutarate. His501 is an additional critical residue at the catalytic site, probably being involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate and the decarboxylation of this cosubstrate.

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Selected References

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  1. Counts D. F., Cardinale G. J., Udenfriend S. Prolyl hydroxylase half reaction: peptidyl prolyl-independent decarboxylation of alpha-ketoglutarate. Proc Natl Acad Sci U S A. 1978 May;75(5):2145–2149. doi: 10.1073/pnas.75.5.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. De Jong L., Kemp A. Stoicheiometry and kinetics of the prolyl 4-hydroxylase partial reaction. Biochim Biophys Acta. 1984 May 31;787(1):105–111. doi: 10.1016/0167-4838(84)90113-4. [DOI] [PubMed] [Google Scholar]
  3. Hanauske-Abel H. M., Günzler V. A stereochemical concept for the catalytic mechanism of prolylhydroxylase: applicability to classification and design of inhibitors. J Theor Biol. 1982 Jan 21;94(2):421–455. doi: 10.1016/0022-5193(82)90320-4. [DOI] [PubMed] [Google Scholar]
  4. Helaakoski T., Annunen P., Vuori K., MacNeil I. A., Pihlajaniemi T., Kivirikko K. I. Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase alpha-subunit isoform: formation of an alpha 2 beta 2 tetramer with the protein disulfide-isomerase/beta subunit. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4427–4431. doi: 10.1073/pnas.92.10.4427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Helaakoski T., Vuori K., Myllylä R., Kivirikko K. I., Pihlajaniemi T. Molecular cloning of the alpha-subunit of human prolyl 4-hydroxylase: the complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4392–4396. doi: 10.1073/pnas.86.12.4392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Jia S., McGinnis K., VanDusen W. J., Burke C. J., Kuo A., Griffin P. R., Sardana M. K., Elliston K. O., Stern A. M., Friedman P. A. A fully active catalytic domain of bovine aspartyl (asparaginyl) beta-hydroxylase expressed in Escherichia coli: characterization and evidence for the identification of an active-site region in vertebrate alpha-ketoglutarate-dependent dioxygenases. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7227–7231. doi: 10.1073/pnas.91.15.7227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jiang F., Peisach J., Ming L. J., Que L., Jr, Chen V. J. Electron spin echo envelope modulation studies of the Cu(II)-substituted derivative of isopenicillin N synthase: a structural and spectroscopic model. Biochemistry. 1991 Dec 3;30(48):11437–11445. doi: 10.1021/bi00112a010. [DOI] [PubMed] [Google Scholar]
  8. Kivirikko K. I., Myllylä R., Pihlajaniemi T. Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J. 1989 Mar;3(5):1609–1617. [PubMed] [Google Scholar]
  9. Kivirikko K. I., Myllylä R. Posttranslational enzymes in the biosynthesis of collagen: intracellular enzymes. Methods Enzymol. 1982;82(Pt A):245–304. doi: 10.1016/0076-6879(82)82067-3. [DOI] [PubMed] [Google Scholar]
  10. Koivu J., Myllylä R., Helaakoski T., Pihlajaniemi T., Tasanen K., Kivirikko K. I. A single polypeptide acts both as the beta subunit of prolyl 4-hydroxylase and as a protein disulfide-isomerase. J Biol Chem. 1987 May 15;262(14):6447–6449. [PubMed] [Google Scholar]
  11. Lamberg A., Pihlajaniemi T., Kivirikko K. I. Site-directed mutagenesis of the alpha subunit of human prolyl 4-hydroxylase. Identification of three histidine residues critical for catalytic activity. J Biol Chem. 1995 Apr 28;270(17):9926–9931. doi: 10.1074/jbc.270.17.9926. [DOI] [PubMed] [Google Scholar]
  12. Majamaa K., Günzler V., Hanauske-Abel H. M., Myllylä R., Kivirikko K. I. Partial identity of the 2-oxoglutarate and ascorbate binding sites of prolyl 4-hydroxylase. J Biol Chem. 1986 Jun 15;261(17):7819–7823. [PubMed] [Google Scholar]
  13. Majamaa K., Hanauske-Abel H. M., Günzler V., Kivirikko K. I. The 2-oxoglutarate binding site of prolyl 4-hydroxylase. Identification of distinct subsites and evidence for 2-oxoglutarate decarboxylation in a ligand reaction at the enzyme-bound ferrous ion. Eur J Biochem. 1984 Jan 16;138(2):239–245. doi: 10.1111/j.1432-1033.1984.tb07907.x. [DOI] [PubMed] [Google Scholar]
  14. McGinnis K., Ku G. M., VanDusen W. J., Fu J., Garsky V., Stern A. M., Friedman P. A. Site-directed mutagenesis of residues in a conserved region of bovine aspartyl (asparaginyl) beta-hydroxylase: evidence that histidine 675 has a role in binding Fe2+. Biochemistry. 1996 Apr 2;35(13):3957–3962. doi: 10.1021/bi951520n. [DOI] [PubMed] [Google Scholar]
  15. Ming L. J., Que L., Jr, Kriauciunas A., Frolik C. A., Chen V. J. NMR studies of the active site of isopenicillin N synthase, a non-heme iron(II) enzyme. Biochemistry. 1991 Dec 17;30(50):11653–11659. doi: 10.1021/bi00114a007. [DOI] [PubMed] [Google Scholar]
  16. Myllylä R., Majamaa K., Günzler V., Hanauske-Abel H. M., Kivirikko K. I. Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase. J Biol Chem. 1984 May 10;259(9):5403–5405. [PubMed] [Google Scholar]
  17. Myllylä R., Pihlajaniemi T., Pajunen L., Turpeenniemi-Hujanen T., Kivirikko K. I. Molecular cloning of chick lysyl hydroxylase. Little homology in primary structure to the two types of subunit of prolyl 4-hydroxylase. J Biol Chem. 1991 Feb 15;266(5):2805–2810. [PubMed] [Google Scholar]
  18. Myllylä R., Tuderman L., Kivirikko K. I. Mechanism of the prolyl hydroxylase reaction. 2. Kinetic analysis of the reaction sequence. Eur J Biochem. 1977 Nov 1;80(2):349–357. doi: 10.1111/j.1432-1033.1977.tb11889.x. [DOI] [PubMed] [Google Scholar]
  19. Pirskanen A., Kaimio A. M., Myllylä R., Kivirikko K. I. Site-directed mutagenesis of human lysyl hydroxylase expressed in insect cells. Identification of histidine residues and an aspartic acid residue critical for catalytic activity. J Biol Chem. 1996 Apr 19;271(16):9398–9402. doi: 10.1074/jbc.271.16.9398. [DOI] [PubMed] [Google Scholar]
  20. Rao N. V., Adams E. Partial reaction of prolyl hydroxylase. (Gly-PRO-Ala)n stimulates alpha-ketoglutarate decarboxylation without prolyl hydroxylation. J Biol Chem. 1978 Sep 25;253(18):6327–6330. [PubMed] [Google Scholar]
  21. Roach P. L., Clifton I. J., Fülöp V., Harlos K., Barton G. J., Hajdu J., Andersson I., Schofield C. J., Baldwin J. E. Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes. Nature. 1995 Jun 22;375(6533):700–704. doi: 10.1038/375700a0. [DOI] [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tuderman L., Myllylä R., Kivirikko K. I. Mechanism of the prolyl hydroxylase reaction. 1. Role of co-substrates. Eur J Biochem. 1977 Nov 1;80(2):341–348. doi: 10.1111/j.1432-1033.1977.tb11888.x. [DOI] [PubMed] [Google Scholar]
  24. Veijola J., Koivunen P., Annunen P., Pihlajaniemi T., Kivirikko K. I. Cloning, baculovirus expression, and characterization of the alpha subunit of prolyl 4-hydroxylase from the nematode Caenorhabditis elegans. This alpha subunit forms an active alpha beta dimer with the human protein disulfide isomerase/beta subunit. J Biol Chem. 1994 Oct 28;269(43):26746–26753. [PubMed] [Google Scholar]
  25. Vuori K., Pihlajaniemi T., Marttila M., Kivirikko K. I. Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7467–7470. doi: 10.1073/pnas.89.16.7467. [DOI] [PMC free article] [PubMed] [Google Scholar]

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