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Biochemical Background

Function of tetrahydrobiopterin

The best established function of tetrahydrobiopterin (BH4) in man is as the natural cofactor for phenylalanine-4-hydroxylase (PAH), tyrosine-3-hydroxylase, and tryptophan-5-hydroxylase; the latter two are key enzymes in the biosynthesis of biogenic amines1.
 

In addition to the hydroxylation of aromatic amino acids, BH4 serves as the cofactor for nitric oxide synthase2 and glyceryl-ether monooxygenase3 (Figure 1).

Aromatic Amino Acid Hydroxylases



     

Figure 1

The function of these reactions derives from the ability of BH4 to react with molecular oxygen to form an active oxygen intermediate that can hydroxylate substrates. In the hydroxylation process, the co-enzyme loses two electrons and is regenerated in vivo in an NADH-dependent reaction. Although BH4 was found to be absolutely essential for nitric oxide synthase activity, the exact function in different forms of the enzyme and the mechanism of action are as yet not clear. Furthermore, it has been demonstrated that BH4 increases the proliferation rate of erythroid cells4 and that it is active as a dopamine releasing factor in rat striatum by using an in vivo dialysis technique5.

Metabolism of tetrahydrobiopterin

BH4 is synthesized from guanosine triphosphate (GTP) in at least four enzymatic steps by the action of three different enzymes (see Figure 1)6-9. GTP cyclohydrolase I (GTPCH), the first enzyme in BH4 biosynthesis, catalyzes the formation of 7,8-dihydroneopterin triphosphate from GTP in a single reaction step10. GTPCH is subject to feed-back inhibition by BH4. Inhibition occurs through a BH4-dependent complex formation between a newly discovered p35 protein and GTPCH11. Furthermore, the inhibition is specifically reversed by phenylalanine. These may explain the high neopterin and biopterin content observed in patients with HPA12.

In the following step, the 6-pyruvoyl-tetrahydropterin synthase (PTPS) catalyzes the conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvoyl-tetrahydropterin13. This conversion is magnesium and zinc dependent and involves the elimination of triphosphate and an intermolecular redox rearrangement reaction14. Whereas GTPCH is found to be highly regulated by the p35 protein, for PTPS no such regulatory processes are described. Nevertheless, PTPS needs posttranslational modification(s), i.e. phosphorylation, in order to be fully active in vivo15. Furthermore, it has been suggested that PTPS is the rate limiting enzyme for BH4 biosynthesis, at least in human liver16

Sepiapterin reductase (SR) has been shown to be an NADPH oxidoreductase required for the final two-step reduction of the diketo intermediate 6-pyruvoyl-tetrahydropterin to BH417.

During the enzymatic hydroxylation of aromatic amino acids, molecular oxygen is consumed and tetrahydrobiopterin is peroxidated and oxidized. The pterin intermediate is subsequently reduced back to BH4 by two enzymes and a reduced pyridine nucleotide (NADH) in a complex recycling reaction (Figure 1). Molecular oxygen is first bound to BH4 to form an unstable 4a-peroxy-tetrahydrobiopterin. The monooxygenation of aromatic amino acids is thus concomitant with oxidation of BH4 to 4a-hydroxy-tetrahydrobiopterin (pterin-4a-carbinolamine)18. Pterin-4a-carbinolamine is subsequently dehydrated to quinonoid-dihydrobiopterin (q-dihydrobiopterin) and water by the specific and highly efficient pterin-4a-carbinolamine dehydratase (PCD)19, 20.



The PCD was originally detected as a contaminant in a preparation of the rat PAH as a consequence of its ability to stimulate the BH4-dependent hydroxylation of phenylalanine21. PCD activity was also shown to be present in human liver, and its absence is concomitant with the formation of 7-substituted pterins22. Unexpectedly, the primary structure of PCD is identical with a protein of the cell nucleus, named dimerization cofactor of hepatocyte nuclear factor 1a (DCoH), reported to have general transcriptional function23-25. However, the evolution of the dual function of regulation of PAH activity and transcription activation in a single protein us unprecedented.

In the last step of BH4 recycling, the q-dihydrobiopterin is reduced back to BH4 by the NADH-dependent dihydropteridine reductase (DHPR). There is no evidence that DHPR has regulatory properties. However, it has been shown that a number of drugs can inhibit the enzyme's activity, both in vivo and in vitro. For instance, methotrexate, an antineoplastic drug commonly used in treatment of cancer, can significantly inhibit DHPR activity26.

REFERENCES

Thöny B, Auerbach G, Blau N. Tetrahydrobiopterin biosynthesis, regeneration, and functions. Biochem J 2000:347:1-16

1. Scriver CR, Eisensmith RC, Woo SLC, Kaufman S. The hyperphenylalaninemias of man and mouse. Annu Rev Genet 1994; 28:141-165.

2. Marletta MA. Nitric oxide synthase structure and mechanism. J. Biol. Chem. 1993; 268:12231-12234.

3. Kaufman S, Pollock RJ, Summer GK, Das AK, Hajra AK. Dependence of an alkyl glycol-ether monooxygenase activity upon tetrahydropterins. Biochim. Biophys. Acta. 1990; 1040:19-27.

4. Tanaka K, Kaufman S, Milstien S. Tetrahydrobiopterin, the cofactor for aromatic amino acid hydroxylases, is synthesized by and regulates proliferation of erythroid cells. Proc. Natl. Acad. Sci. U S A 1989; 86:5864-5867.

5. Koshimura K, Miwa S, Lee K, Fujiwara M, Watanabe Y. Enhancement of dopamine release in vivo from the rat striatum by dialytic perfusion of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin. J. Neurochem. 1990; 54:1391-1397.

6. Nichol CA, Smith GK, Duch DS. Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin. Annu. Rev. Biochem. 1985; 54:729-764.

7. Curtius HC, Heintel D, Ghisla S, Kuster T, Leimbacher W, Niederwieser A. Biosynthesis of tetrahydrobiopterin in man. J. Inherit. Metab. Dis. 1985:28-33.

8. Duch DS, Smith GK. Biosynthesis and function of tetrahydrobiopterin. J. Nutr. Biochem. 1991; 2:411-423.

9. Kaufman S. New tetrahydrobiopterin-dependent systems. Annu. Rev. Nutr. 1993; 13:261-286.

10. Blau N, Niederwieser A. GTP cyclohydrolases: a review. J. Clin. Chem. Clin. Biochem. 1985; 23:169-176.

11. Harada T, Kagamiyama H, Hatakeyama K. Feedback regulation mechanisms for the control of GTP cyclohydrolase I activity. Science 1993; 260:1507-1510.

12. Ponzone A, Guardamagna O, Spada M, Ponzone R, Sartore M, Kierat L, Heizmann CW, Blau N. Hyperphenylalaninemia and pterin metabolism in serum and erythrocytes. Clin. Chim. Acta. 1993; 216:63-71.

13. Takikawa S, Curtius HC, Redweik U, Leimbacher W, Ghisla S. Biosynthesis of tetrahydrobiopterin Purification and characterization of 6-pyruvoyl-tetrahydropterin synthase from human liver. Eur. J. Biochem. 1986; 161:295-302.

14. Bürgisser D, Thöny B, Redweik U, Hess D, Heizmann CW, Huber R, Nar H. 6-Pyruvoyl tetrahydropterin synthase, an enzyme with a novel type of active site involving both zinc binding and an intersubunit catalytic triad motif; Site-directed mutagenesis of the proposed active center, characterization of the metal binding site and modeling of substrate binding. J Mol Biol 1995; 253:358-369.

15. Oppliger T, Thöny B, Nar H, Bürgisser D, Huber R, Heizmann CW, Blau N. Structural and functional consequences of mutations in 6-pyruvoyl tetrahydropterin synthase causing hyperphenylalaninemia in humans. J Biol Chem 1995; 270:1-9.

16. Ichinose H, Ohye T, Matsuda Y, Hori T, Blau N, Burlina A, Rouse B, Matalon R, Fujita K, Nagatsu T. Characterization of mouse and human GTP cyclohydrolase I genes - Mutations in patients with GTP cyclohydrolase I deficiency. J Biol Chem 1995; 270:10062-10071.

17. Smith GK. On the role of sepiapterin reductase in the biosynthesis of tetrahydrobiopterin. Arch. Biochem. Biophys. 1987; 255:254-266.

18. Lazarus RA, Benkovic SJ, Kaufman S. Phenylalanine hydroxylase stimulator protein is a 4a-carbinolamine dehydratase. J Biol Chem 1983; 258:10960-10962.

19. Rebrin I, Bailey SW, Boerth SR, Ardell MD, Ayling JE. Catalytic characterization of 4a-hydroxytetrahydropterin dehydratase. Biochemistry 1995; 34:5801-5810.

20. Köster S, Thöny B, Macheroux P, Curtius HCh, Heitzmann CW, Pfleiderer W, Ghisla S. Human pterin-4a-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1-alpha: characterization and kinetic analysis of wild-type and mutant enzymes. Eur J Biochem 1995; 231:414-423.

21. Kaufman S. A protein that stimulates rat liver phenylalanine hydroxylase. J. Biol. Chem. 1970; 254:4751-4759.

22. Curtius HC, Matasovic A, Schoedon G, Kuster T, Guibaud P, Giudici T, Blau N. 7-Substituted pterins A new class of mammalian pteridines. J. Biol. Chem. 1990; 265:3923-3930.

23. Citron BA, Davis MD, Milstien S, Gutierrez J, Mendel DB, Crabtree GR, Kaufman S. Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins. Proc.. Natl.. Acad.. Sci. USA. 1992; 89:11891-11894.

24. Hauer CR, Rebrin I, Thöny B, Neuheiser F, Curtius HC, Hunziker P, Blau N, Ghisla S, Heizmann CW. Phenylalanine hydroxylase-stimulating protein/pterin-4alpha-carbinolamine dehydratase from rat and human liver - Purification, characterization, and complete amino acid sequence. J. Biol. Chem. 1993; 268:4828-4831.

25. Mendel DB, Khavari PA, Conley PB, Graves MK, Hansen LP, Admon A, Crabtree GR. Characterisation of a cofactor that regulates dimerization of mammalian homeodomain protein. Science 1991; 254:1762-1767.

26. Netter JC, Dhondt JL, Rance F, Petrus M. Early neurotoxicity of high-dose of methotrexate and tetrahydrobiopterin deficiency. Arch. Fr. Pediatr. 1991; 48:719-722.

 


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