The Structural Characterization of 6-Pyruvoyl Tetrahydropterin by Fast Atom Bombardment Mass Spectrometry With One- And Two-Dimensional Mass Analysis (Fab/MS and Fab/Msms)

Richter, Wilhelm J.; Raschdorf, F.; Dahinden, R.; Niederwieser, A.; Curtius, Hans−Christoph; Leimbacher, Walter; Kuster, Thomas

doi:10.1515/9783111503844.39

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“…Due to the lack of well defined fragmentation patterns, the direct FAB/MS method did not allow more precise structural deductions in this case. In particular a differentiation between different isomeric structures of rn 237 Da was not possible (a preliminary attempt towards differentiation by FAB/MSMS has been presented [32]). Sepiapterin and dihydrobiopterin can be ruled out since their chromophore is incompatible with the observed ultraviolet absorption [43].…”

Section: Fabims Studies On the Structure Of Pph4mentioning

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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Section: Fabims Studies On the Structure Of Pph4mentioning

confidence: 99%

¹H‐NMR and mass spectrometric studies of tetrahydropterins

Ghisla

Kuster

Steinerstauch

et al. 1990

European Journal of Biochemistry

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“…OHOH intermediate is 6-pyruvoyl tetrahydropterin (PPH4) whose structure has by now been well defined by our group using NMR and MS techniques ( fig. 1) [5,6],…”

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Tetrahydrobiopterin Biosynthetic Pathway and Deficiency

Niederwieser

Curtius²

1987

Enzyme

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It has been proven that the most common defect in the tetrahydrobiopterin biosynthesis is caused by 6-pyruvoyl tetrahydropterin synthase deficiency. The enzyme 6- pyruvoyl tetrahydropterin synthase consists of four identical subunits which convert dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin in the presence of magnesium. UV, NMR, and MS data prove that the enzyme catalyzes the elimination of triphosphate as well as the intramolecular rearrangement. The 6-pyruvoyl tetrahydropterin synthase activity was measured in fetal erythrocytes and together with the neopterin and biopterin measurements in amniotic fluid this enabled performing prenatal diagnosis of 6-pyruvoyl tetrahydropterin synthase deficiency. Peripheral tetrahydrobiopterin deficiency was shown to be due to an incomplete 6-pyruvoyl tetrahydropterin synthase deficiency or heterozygosity.

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Purification and characterization of 6‐pyruvoyl tetrahydropterin synthase from salmon liver

Hasler

Curtius

1989

European Journal of Biochemistry

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Salmon liver was chosen for the isolation of 6-pyruvoyl tetrahydropterin synthase, one of the enzymes involved in tetrahydrobiopterin biosynthesis. A 9500-fold purification was obtained and the purified enzyme showed two single bands of 16 and 17 kDa on SDS/PAGE. The native enzyme (68 kDa) consists of four subunits and needs free thiol groups for enzymatic activity as was shown by reacting the enzyme with the fluorescent thiol reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The enzyme is heat-stable up to 80 "C, has an isoelectric point of 6.0-6.3, and a pH optimum at 7.5. The enzyme is Mg2+-dependent and has a Michaelis constant for its substrate dihydroneopterin triphosphate of 2.2 pM. The turnover number of the purified salmon liver enzyme is about 50 times as high as that of the enzyme purified from human liver. It does not bind to the lectin concanavalin A, indicating that it is free of mannose and glucose residues.Polyclonal antibodies raised against the purified enzyme in Balb/c mice were able to immunoprecipitate enzyme activity. The same polyclonal serum was not able to immunoprecipitate enzyme activity of human liver 6-pyruvoyl tetrahydropterin synthase, nor was any cross-reaction in ELISA tests seen.

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The Structural Characterization of 6-Pyruvoyl Tetrahydropterin by Fast Atom Bombardment Mass Spectrometry With One- And Two-Dimensional Mass Analysis (Fab/MS and Fab/Msms)

Cited by 3 publications

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¹H‐NMR and mass spectrometric studies of tetrahydropterins

¹H‐NMR and mass spectrometric studies of tetrahydropterins

Tetrahydrobiopterin Biosynthetic Pathway and Deficiency

Purification and characterization of 6‐pyruvoyl tetrahydropterin synthase from salmon liver

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The Structural Characterization of 6-Pyruvoyl Tetrahydropterin by Fast Atom Bombardment Mass Spectrometry With One- And Two-Dimensional Mass Analysis (Fab/MS and Fab/Msms)

Cited by 3 publications

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1H‐NMR and mass spectrometric studies of tetrahydropterins

1H‐NMR and mass spectrometric studies of tetrahydropterins

Tetrahydrobiopterin Biosynthetic Pathway and Deficiency

Purification and characterization of 6‐pyruvoyl tetrahydropterin synthase from salmon liver

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¹H‐NMR and mass spectrometric studies of tetrahydropterins

¹H‐NMR and mass spectrometric studies of tetrahydropterins