The C-6 proton of tetrahydrobiopterin is acquired from water, not NADPH, during de novo biosynthesis.

G K Smith,J A Cichetti,P Chandrasurin,C A Nichol

doi:10.1016/s0021-9258(18)89006-8

Abstract

Tetrahydrobiopterin, the cofactor for the aromatic amino acid hydroxylases, is synthesized in mammals from GTP via a pathway involving both dihydropterin and tetrahydropterin intermediates. In this work, we have investigated the mechanism of conversion of the product formed from GTP, 7,8-dihydroneopterin triphosphate, into the tetrahydropterin intermediates. Tetrahydrobiopterin can be oxidized under conditions which yield pterin or pterin 6-carboxylate without exchange of the C-6 and C-7 protons. Using these techniques, a gas chromatography/mass spectrometry method was developed to determine that in the biosynthesis of tetrahydrobiopterin de novo, in preparations of bovine adrenal medulla, the C-6 proton of tetrahydrobiopterin is derived from water and not from NADPH. In contrast, the C-6 proton of tetrahydrobiopterin produced from sepiapterin (6-lactoyl-7,8-dihydropterin) comes from NADPH. The results are consistent with evidence for the formation of the first tetrahydropterin intermediate by a tautomerization without any requirement for NADPH.

Highlights

GC/MS is an ideal technique for the determination of the incorporation of deuterium into tetrahydrobiopterinsince the 1 mass unit increase which wouldarise couldbe detected inthe mass spectral profile; the possibility that deuterium might be incorporated into the side chain of tetrahydrobiopterinduring biosynthesis might confuse the assignment of the location of any deuterium observed
We chose to remove the tetrahydrobiopterin side chain by the oxidative reactions shown in Fig. 2 [8].The mass spectra of the products of these reactions aremuch simpler, and deuterium incorporation into thepteridine ring cannot be confused with any side chain deuterium
Abundant ions can be seen at m/z 423 [M+]and 408 [M-CH$

Summary

MATERIALS AND METHODS

D20 (99.8%) and 4-amino-3-hydrazino-5-mercapto-l,2,4-tria~le (Received for publication, January 4,1985). The residue was dissolved in 200 ?I of 99.8% D20,15 p l of 8 M NaOD, and 60 pl of saturated KMnOl m 99.8% D20 This solution was incubated for 30 min in the dark a t room temperature after which 10 pl of methanol was added to stop diate to tetrahydrobiopterin in various enzyme systems requires M$+ and NADPH [1,2,3,4]. To obtain a more concentrated enzyme (pterins asa class include those compounds having in common fraction, the DEAE-Sephacel column was eluted with 0.14 the z-amino-4-hydrox~teri~nrieng system); TMS, trimethylsily~ M NaCl in 50 mM Tris-HC1, pH 7.4, rather than theNaCl gradient. Of 50 mM Tris-deuterium chloride, pD 7.6 (99.1% D20)

Enzymatic Reactions

RESULTS

DISCUSSION