Specific interaction of the diastereomers 7(R)‐ and 7(S)‐tetrahydrobiopterin with phenylalanine hydroxylase: implications for understanding primapterinuria and vitiligo

Abstract

Pterin-4a-carbinolamine dehydratase (PCD) is an essential component of the phenylalanine hydroxylase (PAH) system, catalyzing the regeneration of the essential cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin [6(R)BH4]. Mutations in PCD or its deactivation by hydrogen peroxide result in the generation of 7(R,S)BH4, which is a potent inhibitor of PAH that has been implicated in primapterinuria, a variant form of phenylketonuria, and in the skin depigmentation disorder vitiligo. We have synthesized and separated the 7(R) and 7(S) diastereomers confirming their structure by NMR. Both 7(R)- and 7(S)BH4 function as poor cofactors for PAH, whereas only 7(S)BH4 acts as a potent competitive inhibitor vs. 6(R)BH4 (Ki=2.3-4.9 microM). Kinetic and binding studies, as well as characterization of the pterin-enzyme complexes by fluorescence spectroscopy, revealed that the inhibitory effects of 7(R,S)BH4 on PAH are in fact specifically based on 7(S)BH4 binding. The molecular dynamics simulated structures of the pterin-PAH complexes indicate that 7(S)BH4 inhibition is due to its interaction with the polar region at the pterin binding site close to Ser-251, whereas its low efficiency as cofactor is related to a suboptimal positioning toward the catalytic iron. 7(S)BH4 is not an inhibitor for tyrosine hydroxylase (TH) in the physiological range, presumably due to the replacement of Ser-251 by the corresponding Ala297. Taken together, our results identified structural determinants for the specific regulation of PAH and TH by 7(S)BH4, which in turn aid in the understanding of primapterinuria and acute vitiligo.

Identification of pharmacological chaperones as potential therapeutic agents to treat phenylketonuria

“…These K i values are substantially higher than those exhibited by likely physiological inhibitors of PAH, such as 7S-BH 4 (K i ≈ 2-5 μM) (25). Mammalian PAH is regulated by its substrate; the enzyme displays positive cooperativity (Hill coefficient [h] ≈ 2) for l-Phe, and preincubation with the substrate leads to 3-to 4-fold activation (Table 2) (26,27).…”

“…Spectra were corrected for blank emission. Fractional quenching values in the presence of the ligands were calculated from the decrease in fluorescence at 340 nm at increasing ligand concentrations compared with samples without ligand, as reported earlier to determine the binding affinity of different pterins to PAH (25). C0.5 was estimated from fittings to a hyperbolic function, including a linear term for inner filter effects:…”

Queuosine Deficiency in Eukaryotes Compromises Tyrosine Production through Increased Tetrahydrobiopterin Oxidation

“…Numerous studies indicate that BH4 is highly susceptible to auto-oxidation to BH2 in vivo (15,37) and that a low BH4/BH2 ratio, as seen in Qtrt1 gene-trap mice, results in the uncoupling of endothelial NOS leading to superoxide formation and endothelial dysfunction (11,46). Indeed, increased BH4 has proven to be protective in several experimental disease models by its ability to reduce blood pressure (47), decrease atherosclerosis (48), and prevent diabetic complications (49).…”

“…It is known for example that 7-biopterin, formed by spontaneous re-arrangement of 4a-hydroxytetrahydrobiopterin during BH4 recycling, can competitively inhibit the PAH enzyme (37). To rule out this possibility, tyrosine production by cytosolic liver extracts diluted only marginally (1:20) by the addition of buffer, catalase (170 ng/l), and (6R)-BH4 cofactor (100 M) was performed (Fig.…”

Melanogenesis Inhibition Due to NADH

“…40) This system, under normal physiological conditions, is always reduced by NADH, so that this coenzyme is attacked by TYR and contribute to the generation of H 2 O 2 in processes such as vitiligo. 41,42) Moreover, consumption of NADH hinder the action of the NADH-dependent dihydropteridine reductase, which reduces quinonoid dihydrobiopterin to 6(R)-L-erytro-5,6,7,8-tetrahydrobiopterin (BH 4 ).…”