Dataset Information

Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations.

ABSTRACT: Phenylketonuria patients harboring a subset of phenylalanine hydroxylase (PAH) mutations have recently shown normalization of blood phenylalanine levels upon oral administration of the PAH cofactor tetrahydrobiopterin [(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4)]. Several hypotheses have been put forward to explain BH4 responsiveness, but the molecular basis for the corrective effect(s) of BH4 has not been understood. We have investigated the biochemical, kinetic, and structural changes associated with BH4-responsive mutations (F39L, I65T, R68S, H170D, E178G, V190A, R261Q, A300S, L308F, A313T, A373T, V388M, E390G, P407S, and Y414C). The biochemical and kinetic characterization of the 15 mutants studied points toward a multifactorial basis for the BH4 responsiveness; the mutants show residual activity (>30% of WT) and display various kinetic defects, including increased Km (BH4) and reduced cooperativity of substrate binding, but no decoupling of cofactor (BH4) oxidation. For some, BH4 seems to function through stabilization and protection of the enzyme from inactivation and proteolytic degradation. In the crystal structures of a phenylketonuria mutant, A313T, minor changes were seen when compared with the WT PAH structures, consistent with the mild effects the mutant has upon activity of the enzyme both in vitro and in vivo. Truncations made in the A313T mutant PAH form revealed that the N and C termini of the enzyme influence active site binding. Of fundamental importance is the observation that BH4 appears to increase Phe catabolism if at least one of the two heterozygous mutations has any residual activity remaining.

SUBMITTER: Erlandsen H

PROVIDER: S-EPMC534739 | biostudies-literature | 2004 Nov

REPOSITORIES: biostudies-literature

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Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations.

Erlandsen Heidi H Pey Angel L AL Gámez Alejandra A Pérez Belén B Desviat Lourdes R LR Aguado Cristina C Koch Richard R Surendran Sankar S Tyring Stephen S Matalon Reuben R Scriver Charles R CR Ugarte Magdalena M Martínez Aurora A Stevens Raymond C RC

Proceedings of the National Academy of Sciences of the United States of America 20041119 48

Phenylketonuria patients harboring a subset of phenylalanine hydroxylase (PAH) mutations have recently shown normalization of blood phenylalanine levels upon oral administration of the PAH cofactor tetrahydrobiopterin [(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4)]. Several hypotheses have been put forward to explain BH4 responsiveness, but the molecular basis for the corrective effect(s) of BH4 has not been understood. We have investigated the biochemical, kinetic, and structural changes ass ...[more]

PMID: 15557004

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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:Tetrahydrobiopterin (BH(4)) responsiveness is currently defined as a decrease in plasma phenylalanine concentrations in patients with phenylketonuria (PKU). This definition does not offer insight beyond the initial assessment of patients, which may lead to treatment ambiguity in patients who only experience an initial decrease in plasma phenylalanine concentrations. We present our experience with a novel classification approach using sequentially-applied criteria. Plasma phenylalanine concentrations were measured at baseline and after one month of BH(4) therapy (20 mg/kg/day) in 58 PKU patients (34 M, 24 F; age 17.3±11.0 years). Thirty-two patients (55.2%) were classified as "preliminary responders" at one month, experiencing at least a 15% decrease in plasma phenylalanine concentrations. Preliminary responders' ability to liberalize their dietary restrictions was then systematically assessed. "Definitive responders" were defined as preliminary responders who could increase their dietary phenylalanine tolerance by at least 300 mg/day and lower prescribed medical food needs by at least 25% while maintaining metabolic control (plasma phenylalanine ?360 ?mol/L) and consuming adequate dietary protein. Preliminary responders who could not liberalize their diets according to these criteria were classified as "provisional responders." Nineteen patients (32.8% of patients initiating BH(4) therapy) met the definitive responder criteria, increasing dietary phenylalanine tolerance from 704±518 mg/day to 1922±612 mg/day and reducing medical food to 16.7±19.5% of their baseline prescription. Nine patients (15.5% of patients initiating BH(4) therapy) were classified as provisional responders, all remaining on 100% of their baseline medical food prescription. From this classification approach, a subgroup of provisionally responsive patients emerged who experienced an initial decrease in plasma phenylalanine concentrations but who could not substantially increase their dietary phenylalanine tolerance or decrease medical food needs. Diet liberalization is an essential component of BH(4)-responsiveness classification.

Dataset Information

Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations.

Publications

Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations.

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