The biosynthesis oftetrahydrobiopterin (BH 4 ) from dihydroneopterin triphosphate (NH lP 3 ) was studied in human liver extract. The phosphate-eliminating enzyme (PEE) was purified ~ 750-fold. The conversion of NH lP, to BH 4 was catalyzed by this enzyme in the presence of partially purified sepiapterin reductase. Mg1 -and NADPH. The PEE is heat stable when heated at 80 "C for 5 min. It has a molecular weight of 63 OOU daltons. One possible intermediate 6-( I'-hydroxy-2 '-oxopropyl )5.6. 7.8-tetrahydropterin( 2' -oxo-tetrahydropterin I was formed upon incubation of BH 4 in the presence of sepiapterin reductase and NADP T at pH 9.0. Reduction of this compound with NaBD 4 yielded monodeutero threo and erythro-BH 4 • the deuterium was incorporated at the 2' position. This and the UV spectra were consistent with a 2'-oxo-tetrahydropterin structure. Dihydrofolate reductase (DHFR I catalyzed the reduction of BH l to BH 4 and was found to be specific for the pro-R-NADPH side. The sepiapterin reductase catalyzed the transfer of the pro-S hydrogen of • NAD PH during the reduction of sepiapterin to BH 1. In the presence of crude liver extracts the conversion of NH1P, to BH 4 requires NADPH. Two deuterium atoms were incorporated from (4S-1 H INADHP in the I' and 2' position of the BH 4 side chain. Incorporation of one hydrogen from the solvent was found at position C(6). These results are consistent with the occurrence of an intramolecular redox exchange between the pteridine nucleus and the side chain and formation of 6-pyruvoyl-5.6,7,8-tetrahydropterin(tetrahydro-1 '-2 '-dioxopterin) as intermediate.

The biosynthesis of tetrahydrobiopterin from either dihydroneopterin triphosphate, sepiapterin, dihydrosepiapterin or dihydrobiopterin was investigated using extracts from human liver, dihydrofolate reductase and purified sepiapterin reductase from human liver and rat erythrocytes. The incorporation of hydrogen in tetrahydrobiopterin was studied in either 2H2O or in H2O using unlabeled NAD(P)H or (R)‐(4‐2H)NAD(P)H or (S)‐(4‐2H)NAD(P)H. Dihydrofolate reductase catalyzed the transfer of the pro‐R hydrogen of NAD(P)H during the reduction of 7,8‐dihydrobiopterin to tetrahydrobiopterin. Sepiapterin reductase catalyzed the transfer of the pro‐S hydrogen of NADPH during the reduction of sepiapterin to 7,8‐dihydrobiopterin. In the presence of partially purified human liver extracts one hydrogen from the solvent is introduced at position C(6) and the 4‐pro‐S hydrogen from NADPH is incorporated at each of the C(1′) and C(2′) position of BH4. Label from the solvent is also introduced into position C(3′). These results suggest that dihydrofolate reductase is not involved in the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. They are consistent with the assumption of the occurrence of a 6‐pyruvoyl‐tetrahydropterin intermediate, which is proposed to be formed upon triphosphate elimination from dihydroneopterin triphosphate, and via an intramolecular redox reaction. Our results suggest that the reduction of 6‐pyruvoyl‐tetrahydropterin might be catalyzed by sepiapterin reductase.

-The biosynthetic pathway of tetrahydrobiopterin (BH 4 ) from dihydroneopterin triphosphate (NH 2 P 3 ) was studied in fresh as well as heat-treated human liver extracts. The question of NAD(P)H dependency for the formation of sepiapterin was examined. NH 2 P 3 was converted by fresh extracts to sepiapterin in low quantities (2% conversion) in the absence of exogenously added NADPH as well as under conditions that ensured the destruction of endogenous, free NAD(P)H. The addition of NADPH to the fresh liver extracts stimulated the synthesis of BH 4 to a much higher yield (17% conversion), and the amount of sepiapterin formed was reduced to barely detectable levels. In contrast, the heat-treated extract (enzyme A2 fraction) formed sepiapterin (1.3% conversion) only in the presence and not in the absence of NADPH. These results indicate that sepiapterin may not be an intermediate on the pathway leading to BH 4 biosynthesis under normal~vivo conditions. Rather, sepiapterin may result from the breakdown of an as yet unidentified intermediate that is actually on the pathway. It is speculated that NH 2 P 3 may be converted to a diketo-tetrahydropterin intermediate (or an equivalent tautomeric structure) by a mechanism involving an intramolecular oxidoreduction reaction. A diketo-tetrahydropterin intermediate could be converted to 5,6-dihydrosepiapterin, which also has a tetrahydropterin ring system and can be converted directly to BH 4 by sepiapterin reductase. This proposed pathway can explain how the tetrahydropterin ring system can be formed without sepiapterin, dihydrobiopterin, or dihydrofolate reductase being involved in BH 4 biosynthesis~vivo.

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The biosynthesis oftetrahydrobiopterin (BH 4 ) from dihydroneopterin triphosphate (NH lP 3 ) was studied in human liver extract. The phosphate-eliminating enzyme (PEE) was purified ~ 750-fold. The conversion of NH lP, to BH 4 was catalyzed by this enzyme in the presence of partially purified sepiapterin reductase. Mg1 -and NADPH. The PEE is heat stable when heated at 80 "C for 5 min. It has a molecular weight of 63 OOU daltons. One possible intermediate 6-( I'-hydroxy-2 '-oxopropyl )5.6. 7.8-tetrahydropterin( 2' -oxo-tetrahydropterin I was formed upon incubation of BH 4 in the presence of sepiapterin reductase and NADP T at pH 9.0. Reduction of this compound with NaBD 4 yielded monodeutero threo and erythro-BH 4 • the deuterium was incorporated at the 2' position. This and the UV spectra were consistent with a 2'-oxo-tetrahydropterin structure. Dihydrofolate reductase (DHFR I catalyzed the reduction of BH l to BH 4 and was found to be specific for the pro-R-NADPH side. The sepiapterin reductase catalyzed the transfer of the pro-S hydrogen of • NAD PH during the reduction of sepiapterin to BH 1. In the presence of crude liver extracts the conversion of NH1P, to BH 4 requires NADPH. Two deuterium atoms were incorporated from (4S-1 H INADHP in the I' and 2' position of the BH 4 side chain. Incorporation of one hydrogen from the solvent was found at position C(6). These results are consistent with the occurrence of an intramolecular redox exchange between the pteridine nucleus and the side chain and formation of 6-pyruvoyl-5.6,7,8-tetrahydropterin(tetrahydro-1 '-2 '-dioxopterin) as intermediate.