Purification and properties of the phosphate eliminating enzyme involved in the biosynthesis of BH4 in man

Heintel, Dorothee; Leimbacher, Walter; Redweik, U.; Zagalak, B.; Curtius, Hans−Christoph

doi:10.1016/s0006-291x(85)80146-7

Cited by 27 publications

(13 citation statements)

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“…The results presented in this paper indicate that PPH4S is a single protein which catalyzes two reactions, that is, elimination of phosphate from NHzTP ( I ) [33] and an internal oxidoreduction leading to PPH4 (4) (Scheme 1). The enzyme consists of four identical subunits of 19 kDa each, the molecular mass of tetrameric PPH4S being 83 kDa.…”

Section: Discussionmentioning

confidence: 82%

“…The experimental determination of the sequence of these two steps, i.e. elimination of triphosphate [33] and the internal oxidoreduction, might prove very difficult to achieve. It is conceivable that the same enzyme base which is assumed to induce triphosphate elimination by abstracting the 2'-hydrogen as a proton might be positioned to remove also the 1'-H (as a proton) and thus to catalyze also the internal redox shuttle (Scheme 1).…”

Section: Discussionmentioning

confidence: 99%

“…Assay for PPH4S activity PPH4S activity was measured based on the observation that BH4 is produced from NHzTP by PPH4S and sepiapterin reductase in the presence of Mg2+ and NADPH [30,33,341. The amount of BH4 produced was measured by HPLC with electrochemical detection [36].…”

Section: Methodsmentioning

confidence: 99%

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Biosynthesis of tetrahydrobiopterin. Purification and characterization of 6-pyruvoyl-tertrahydropterin synthase from human liver

et al. 1986

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6-Pyruvoyl-tetrahydropterin synthase, which catalyzes the first step in the conversion of 7,s-dihydroneopterin triphosphate to tetrahydrobiopterin, was purified approximately 140,000-fold to apparent homogeneity from human liver. The molecular mass of the enzyme is estimated to be 83 kDa. 7,X-Dihydroneopterin triphosphate was a substrate of the enzyme in the presence of Mg2+, and the pH optimum of the reaction was 7.5 in Tris HCI buffer. The K, value for 7,fLdihydroneopterin triphosphate was 10 pM. The product of this enzymatic reaction was the presumed intermediate 6-pyruvoyl-tetrahydropterin. This latter compound was converted to tetrahydrobiopterin in the presence of NADPH and partially purified sepiapterin reductase from human liver. The conditions and the effect of N-acetylserotonin on this reaction, and on the formation of the intermediates 6-(1 '-hydroxy-2'-oxopropyl)-tetrahydropterin and 6-( 1'0x0-2'-hydroxypropy1)-tetrahydropterin have been studied.

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Section: Discussionmentioning

confidence: 82%

Section: Discussionmentioning

confidence: 99%

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Biosynthesis of tetrahydrobiopterin. Purification and characterization of 6-pyruvoyl-tertrahydropterin synthase from human liver

et al. 1986

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“…The assay for PTS activity in human PBM is based on that developed for human liver [19]. The reaction mixture consisted of 100 mM Tris/HCl, pH 7.4, 5 mM dithioerythritol, 25 pM NH2TP, 8 mM MgCI2, 1 mM NADPH, approximately 2 mU SR, and cell lysate, in a final volume of 125 pl.…”

Section: Activity Of 6-pyruvoyl Tetrahydropterin Synthasementioning

confidence: 99%

Biosynthesis and metabolism of pterins in peripheral blood mononuclear cells and leukemia lines of man and mouse

et al. 1987

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The cellular origin and the control of neopterin release associated with immune stimulation was studied in cell cultures. Using purified human mononuclear cells, the intracellular change in concentrations of GTP and pterins was measured under various kinds of stimulation. Three enzymes involved in tetrahydrobiopterin biosynthesis, i.e. GTP cyclohydrolase I, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase, were also determined.Human macrophages stimulated with culture supernatant from activated T-lymphocytes were the main producers of neopterin. In these cells, GTP cyclohydrolase I activity was elevated due to high GTP levels and therefore neopterin accumulated. Human macrophages lack 6-pyruvoyl tetrahydropterin synthase activity. Exogenous tetrahydrobiopterin added to the culture medium of stimulated T cells and macrophages suppressed the elevation of GTP cyclohydrolase I activity and neopterin concentration, but not the elevation of intracellular GTP.Stimulation of macrophages with recombinant human interferon-y and neutralization of the effect of T cell supernatants by addition of a monoclonal antibody specific for human interferon-y showed that immune interferon induced the alterations in GTP cyclohydrolase I activity and neopterin concentration. In the human macrophage line U-937 and in the leukemia line HL-60, no GTP cyclohydrolase I activity or intracellular pterins were detected, but high levels of GTP. In mouse mononuclear cells, no neopterin was detected, but biopterin and pterin. After stimulation, biopterin was elevated in the same way as neopterin in human mononuclear cells. This is explained by the different regulation of the rate-limiting steps of tetrahydrobiopterin biosynthesis in man and in mouse. These results suggest that neopterin is an unspecific marker for the activation of the cellular immune system.

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“…While the chemical structures and some of the properties of these intermediates are known, the mechanisms by which 7,s-dihydroneopterin triphosphate (NH2TP) and 6-pyruvoyl-5,6,7,8-tetrahydropterin (PPH4) are formed have not yet been elucidated. The first step, the conversion of GTP to NH2TP, is an apparently rather complicated process catalyzed by a single enzyme, GTP cyclohydrolase I, and involving ring opening of GTP, extrusion of a formyl equivalent, rearrangement of the ribosyl moiety and reclosure to form a six-membered ring [19, 201. The second step also involves two chemically distinct processes, the elimination of triphosphate from the terminal (C3') position of the C(6) side chain (23, 21 -231, and an internal, Amadori-type rearrangement during which the N(5)-C(6) double bond of NH2TP is reduced at the expense of oxidation of C(l')-OH to C(l')=O 13,12,21,[23][24][25][26] A further major issue at the onset of our work was the chemical identity of PPH, and its properties. The intermediacy of this molecule in BH, biosynthesis had been proposed by several groups 16, 7, 23 -251, but its high instability and the difficulties in obtaining substantial quantities of material have hindered structural studies.…”

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confidence: 99%

¹H‐NMR and mass spectrometric studies of tetrahydropterins

Ghisla

Kuster

Steinerstauch

et al. 1990

European Journal of Biochemistry

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The conversion of dihydroneopterin triphosphate in the presence of 6-pyruvoyl tetrahydropterin synthase was followed by 'H-NMR spectroscopy. The interpretation of the spectra of the product is unequivocal: they show formation of a tetrahydropterin system carrying a stereospecifically oriented substituent at the asymmetric C(6) atom. The spectra are compatible with formation of a (3')-CH3 function, and with complete removal of the 1' and 2' hydrogens of dihydroneopterin triphosphate. The fast-atom-bombardment/mass spectrometry study of the same product yields a [M+H]+ ion at m/z 238 compatible with the structure of 6-pyruvoyl tetrahydropterin. The data support the proposed structure of 6-pyruvoyl tetrahydropterin as a key intermediate in the biosynthesis of tetrah ydrobiopterin.The biosynthetic pathway of tetrahydrobiopterin (BH,) has been elucidated in its prominent features in the past few years by the convergent efforts of several groups [I -91. The following sequence is now being widely accepted: GTP 4 7,X-dihydroneopterin triphosphate (NH2TP) + h-pyruvoyl-5,6,7,%tetrahydropterin (PPH,) 6-lactoyl-5,6,7,8-tetrahydropterin or 6-hydroxy-acetonoyl-5,6,7,X-tetrahydropterin + tetrahydrobiopterin (BH,).The enzymes involved have also been purified and characterized 14, 10-281. While the chemical structures and some of the properties of these intermediates are known, the mechanisms by which 7,s-dihydroneopterin triphosphate (NH2TP) and 6-pyruvoyl-5,6,7,8-tetrahydropterin (PPH4) are formed have not yet been elucidated. The first step, the conversion of GTP to NH2TP, is an apparently rather complicated process catalyzed by a single enzyme, GTP cyclohydrolase I, and involving ring opening of GTP, extrusion of a formyl equivalent, rearrangement of the ribosyl moiety and reclosure to form a six-membered ring [19, 201. The second step also involves two chemically distinct processes, the elimination of triphosphate from the terminal (C3') position of the C(6) side chain (23, 21 -231, and an internal, Amadori-type rearrangement during which the N(5)-C(6) double bond of NH2TP is reduced at the expense of oxidation of C(l')-OH to C(l')=O 13, 12,21,[23][24][25][26] A further major issue at the onset of our work was the chemical identity of PPH, and its properties. The intermediacy of this molecule in BH, biosynthesis had been proposed by several groups 16, 7, 23 -251, but its high instability and the difficulties in obtaining substantial quantities of material have hindered structural studies. In fact, the only feasible procedure for its preparation requires purified 6-pyruvoyl tetrahydropterin synthase and purified NH2TP [27], the latter also having to be prepared enzymatically from GTP [28]. Furthermore, the evidence presented for its structure was deduced from trapping experiments involving modifications of the pyruvoyl function such as reduction with NaBH4 or enzymatically with NADPH to BH4 [3, 4, 6, 8, 22, 23, 25, 26, 291. In the present work we report detailed 'H-NMR and fastatom-bombardment/mass spectrometry (FAB/MS) ...

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Purification and properties of the phosphate eliminating enzyme involved in the biosynthesis of BH4 in man

Cited by 27 publications

References 12 publications

Biosynthesis of tetrahydrobiopterin. Purification and characterization of 6-pyruvoyl-tertrahydropterin synthase from human liver

Biosynthesis of tetrahydrobiopterin. Purification and characterization of 6-pyruvoyl-tertrahydropterin synthase from human liver

Biosynthesis and metabolism of pterins in peripheral blood mononuclear cells and leukemia lines of man and mouse

¹H‐NMR and mass spectrometric studies of tetrahydropterins

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Purification and properties of the phosphate eliminating enzyme involved in the biosynthesis of BH4 in man

Cited by 27 publications

References 12 publications

Biosynthesis of tetrahydrobiopterin. Purification and characterization of 6-pyruvoyl-tertrahydropterin synthase from human liver

Biosynthesis of tetrahydrobiopterin. Purification and characterization of 6-pyruvoyl-tertrahydropterin synthase from human liver

Biosynthesis and metabolism of pterins in peripheral blood mononuclear cells and leukemia lines of man and mouse

1H‐NMR and mass spectrometric studies of tetrahydropterins

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¹H‐NMR and mass spectrometric studies of tetrahydropterins