The auto‐oxidation of tetrahydrobiopterin

Abstract

The product of the aerobic oxidation of tetrahydrobiopterin, quinonoid dihydrobiopterin, is unstable and rapidly rearranges to form a 7,s-dihydropteridine. Kaufman [Kaufman, S. (1967) J. Biol. Chem. 242,3934-39431 identified the stable product produced in 0.1 M phosphate pH 6.8, as 7,s-dihydrobiopterin. However, Armarego et al. [Armarego, W. L. F., Randles, D. and Taguchi, H. (1983) Eur. J. Biochem. 135 393-4031 questioned this assignment because they found that the dihydroxypropyl group on C-6 was eliminated and 7,s-dihydropterin was the predominant product when the aerobic oxidation was performed in 0.1 M Tris pH 7.6. In the present study we demonstrate that the rearrangement of the unstable quinonoid dihydrobiopterin results in a mixture of these two 7,8-dihydropteridines at neutral pH, 25 "C. Furthermore, we find that the loss or retention of the alkyl sidechain is not solely dependent on the pH of the reaction mixture, as was previously assumed by Armarego et al., but rather is strongly influenced by the temperature and the type of buffer. In addition, we describe a new method for quantifying the relative amounts of these two 7,8-dihydropteridines in mixtures of unknown concentrations. This method relies on multicomponent analysis of second derivative spectra and results in values which agree with the concentrations determined directly by HPLC.A reduced pteridine cofactor is necessary for phenylalanine hydroxylase to catalyze the conversion of phenylalanine to tyrosine [l]. The natural cofactor for this enzyme (as well as for tyrosine hydroxylase [2-51 and tryptophan hydroxylase [6, 71 is tetrahydrobiopterin, (BH,) [S]. During the hydroxylation reaction at neutral pH, there is a stoichiometric oxidation of BH4 to quinonoid dihydrobiopterin (qBH2) [9, 101. This latter species can then be reduced back to BH4 by the NADH-dependent enzyme, dihydropteridine reductase [ l l -151. qBH2 is unstable and, in the absence of the reductase, rapidly rearranges to a 7,s-dihydropteridine [9]. It was concluded that 7,s-dihydrobiopterin (BH,) was the product of the tautomerization of qBH2 [16,17] because when the auto-oxidation of BH4 was performed in 0.1 M phosphate pH 6.8 at ambient temperature, the ultraviolet spectrum of the product was identical to that of authentic BH2 [16]. This conclusion was also strongly supported by the finding that one of the products of the aerobic oxidation of BH4 in phosphate buffer was a substrate for sepiapterin reductase, an enzyme that catalyzes the NADP-dependent oxidation of the 2'-hydroxyl group on the side-chain of BH2 [16].However, it has become apparent that the unstable qBH2 does not always isomerize to BH,. Whereas the oxidation of Correspondence to

Kinetics of acid-induced degradation of tetra- and dihydrobiopterin in relation to their relevance as biomarkers of endothelial function

“…As shown here, BH 2 is only degraded to a small extent in 30 min. Davis et al observed no degradation of BH 2 for up to 150 min at pH 6.8-8.2 (Davis et al, 1988), which is in line with our results showing little degradation during this short-time span. On the other hand, Dántola et al followed the degradation of BH 2 at neutral pH at 25°C for 2 days and observed very little degradation (Dántola et al, 2008).…”

“…The half-life of BH 4 can be estimated to be around half an hour at pH 3 and at pH 4 and above almost complete oxidation takes place in 20 min (Lyudnikova et al, 2009). At pH 5.3-7.6 the half-life was estimated to around 9 min (Heales et al, 1988), and another study found half-lives of 6.5-60 min at pH 6.8-8.2 depending on the buffer used (Davis et al, 1988). Similarly, a half-life of 15-20 min was observed at pH 7.4 (Valent & Tóth, 2006).…”

Alteration of striatal tetrahydrobiopterin in iron-induced unilateral model of Parkinson's disease

“…All the monoaminergic neurons and terminals in the CNS produce BH4 and DA cells are vulnerable to high level of BH4, most probably due to the higher rate of oxidation of DA compared with other catecholamine or serotonin [18,19]. Under, the normal conditions, the level of GTP-CH I expression in the nigrostriatal system is lower than in the other monoaminergic cells [23,24].…”

“…BH4 auto-oxidation could produce hydrogen peroxide and superoxide radical [18][19][20][21]. In addition, DA, with its synthesis facilitated by BH4, in turn undergoes auto-oxidation to form a reactive quinine species [18,22,24].…”

“…It has been demonstrated that BH4 and DA together lead to generation of oxidative stress and render DA cells particularly vulnerable [16] and furthermore administration of BH4 to animals leads to as selective degeneration of the DA system [17]. BH4 itself generates oxidative stress by undergoing auto-oxidation [18][19][20][21] and elicits the formation of reactive quinonoid derivatives of DA and protein as well as lipid peroxidation in DA cells [22]. The amount of BH4 synthesizing enzyme, GTP cyclohydrolase I (GTP-CH I), is lowest, suggesting that the level of BH4 in this area might be normally subtoxic [23,24].…”