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Contrasting effects of N5-substituted tetrahydrobiopterin derivatives on phenylalanine hydroxylase, dihydropteridine reductase and nitric oxide synthase.

ABSTRACT: Tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H(4)biopterin] is one of several cofactors of nitric oxide synthases (EC 1.14.13.39). Here we compared the action of N(5)-substituted derivatives on recombinant rat neuronal nitric oxide synthase with their effects on dihydropteridine reductase (EC 1.6.99.7) and phenylalanine hydroxylase (EC 1.14.16.1),the well-studied classical H(4)biopterin-dependent reactions. H(4)biopterin substituted at N(5) with methyl, hydroxymethyl, formyl and acetyl groups were used. Substitution at N(5) occurs at a position critical to the redox cycle of the cofactor in phenylalanine hydroxylase/dihydropteridine reductase. We also included N(2)'-methyl H(4)biopterin, a derivative substituted at a position not directly involved in redox cycling, as a control. As compared with N(5)-methyl H(4)biopterin, N(5)-formyl H(4)biopterin bound with twice the capacity but stimulated nitric oxide synthase to a lesser extent. Depending on the substituent used, N(5)-substituted derivatives were redox-active: N(5)-methyl- and N(5)-hydroxyl methyl H(4)biopterin, but not N(5)-formyl- and N(5)-acetyl H(4)biopterin, reduced 2,6-dichlorophenol indophenol. N(5)-Substituted H(4)biopterin derivatives were not oxidized to products serving as substrates for dihydropteridine reductase and,depending on the substituent, were competitive inhibitors of phenylalanine hydroxylase: N(5)-methyl- and N(5)-hydroxymethyl H(4)biopterin inhibited phenylalanine hydroxylase, whereas N(5)-formyl- and N(5)-acetyl H(4)biopterin had no effect. Our data demonstrate differences in the mechanism of stimulation of phenylalanine hydroxylase and nitric oxide synthase by H(4)biopterin. They are compatible with a novel, non-classical, redox-active contribution of H(4)biopterin to the catalysis of the nitric oxide synthase reaction.

SUBMITTER: Werner ER

PROVIDER: S-EPMC1221100 | biostudies-other | 2000 Jun

REPOSITORIES: biostudies-other

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Project description:A need exists to expand the characterization of tetrahydrobiopterin (BH(4)) responsiveness in patients with phenylketonuria (PKU), beyond simply evaluating change in blood phenylalanine concentrations. The clinical interpretation of BH(4) responsiveness should be evaluated within the context of phenylalanine hydroxylase (PAH) genotype.This investigation seeks to use a modified version of a previously developed PAH genotype severity tool, the assigned value (AV) sum, to assess the molecular basis of responsiveness in a clinical cohort and to explore the tool's ability to differentiate BH(4) responsive groups.BH(4) response was previously clinically classified in 58 patients with PKU, with three response groups emerging: definitive responders, provisional responders, and non-responders. Provisional responders represented a clinically ambiguous group, with an initial decrease in plasma phenylalanine concentrations, but limited ability to improve dietary phenylalanine tolerance. In this retrospective analysis, mutations in the PAH gene were identified in each patient. PAH genotype was characterized through the AV sum approach, in which each mutation is given an AV of 1, 2, 4, or 8; the sum of both mutations' AV corresponds to genotype severity, with a lower number representing a more severe phenotype. An AV sum cutoff of 2 (indicative of the most severe genotypes) was used to dichotomize patients and predict BH(4) responsiveness. Provisional responders were classified with the definitive responders then the non-responders to see with which group they best aligned.In 17/19 definitive responders, at least one mutation was mild or moderate in severity (AV sum>2). In contrast, 7/9 provisional responders carried two severe or null mutations (AV sum=2), suggesting little molecular basis for responsiveness. Non-responders represent a heterogeneous group with 15/25 patients carrying two severe mutations (AV sum=2), 5/25 patients carrying one moderate or mild mutation in combination with a severe or null mutation (AV sum>2), and the remaining five patients carrying an uncharacterized mutation in combination with a severe mutation. Predictive sensitivity of the AV sum was maximized (89.5% vs. 67.9%) with limited detriment to specificity (79.4% vs. 80.0%), by classifying provisional responders with the non-responders rather than with the definitive responders.In our clinical cohort, the AV sum tool was able to identify definitive responders with a high degree of sensitivity. As demonstrated by both the provisional responder group and the substantial number of non-responders with AV sums>2, a potential exists for misclassification when BH(4) response is determined by relying solely on change in plasma phenylalanine concentrations. PAH genotype should be incorporated in the clinical evaluation of BH(4) responsiveness.

Project description:BackgroundInherited phenylalanine hydroxylase deficiency, also known as phenylketonuria (PKU), causes poor growth and neurologic deficits in the untreated state. After ascertainment through newborn screen and dietary phenylalanine (Phe) restriction to achieve plasma Phe in the range of 120-360 μmol/L, these disease manifestations can be prevented. Poor compliance with protein restricted diets supported by medical food is typical in later years, beginning in the late toddler and teenage years. Pharmacologic doses of oral tetrahydrobiopterin (BH4; sapropterin dihydrochloride) is effective in reducing plasma Phe in about 40-50% of PKU patients but effectiveness is highly variable.ObjectiveTo assess the maximal responsiveness to 20 mg/kg/day oral BH4 as it affects plasma Phe and dietary Phe allowance in PKU patients.Materials and methodsThis was a single-center, retrospective observational study, combining case reports of individual patients. We reported an outcome of 85 patients with PKU who were trialed on BH4. Phe levels and dietary records of 19 BH4 "super-responders" were analyzed.ResultsOverall, 63.5% of the patients (54/85) were considered BH4 responders. However, we quantitated the dietary liberalization of 19 of our responsive patients (35%), those with at least a 2-fold increase in dietary Phe and maintenance of plasma Phe in treatment range. In these "super-responders", the mean plasma Phe at baseline was 371 ± 237 μmol/L and decreased to 284 ± 273 μmol/L after 1 year on BH4. Mean dietary Phe tolerance increased significantly from 595 ± 256 to 2260 ± 1414 mg/day (p ≤0.0001), while maintaining mean plasma Phe levels within treatment range. Four patients no longer required dietary Phe restriction and could discontinue medical food. The majority of patients had at least one BH4-responsive genotype.ConclusionThis cohort demonstrates the maximally achievable dietary liberalization which some PKU patients may expect with BH4 therapy. Health benefits are considered to accrue in patients with increased intact protein.

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Contrasting effects of N5-substituted tetrahydrobiopterin derivatives on phenylalanine hydroxylase, dihydropteridine reductase and nitric oxide synthase.

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