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 BH 4 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.

Chaperone-Like Therapy with Tetrahydrobiopterin in Clinical Trials for Phenylketonuria: Is Genotype a Predictor of Response?

“…1b). Because our study is not a comprehensive sample of the human PKU/HPA population, a limitation compounded by a relaxed protocol design, we could not readily reveal the true prevalence of responsive mutations; for example, E390G in the catalytic domain is strongly associated with a chaperone-like effect (Erlandsen et al 2004;Pey et al 2004) yet did not readily disclose this property here.…”

“…The responsive mutations seldom mapped to the cofactor binding site. One responsive allele, p.Y414C, maps to the dimer-dimer interfaces of the tetramer, suggesting that sapropterin enhances stabilization of the tetramer (Erlandsen et al 2004;Pey et al 2004). Among the other responsive mutations, p.I65T and p.A104D are located in the regulatory domain, while p.Q226H, p.R241H, and p.A309D are part of the catalytic domain (Fig.…”

“…The metabolic responsiveness of our subset of sapropterin-responsive HPA/PKU patients is explained by a different process, namely enhancement of residual PAH enzyme activity and opening of the oxidative pathway (Muntau et al 2002). Pharmacological doses of sapropterin appear to achieve this effect, mainly in association with PAH missense alleles, either by a kinetic effect to overcome unfavorable binding of cofactor, or a chaperone-like effect on a misfolding enzyme subunit (Bechtluft et al 2007; Dobrowolski et al a These genotypes were classified as unresponsive based on findings of PKU-001 and PKU-003 and/or PKU-004 b This genotype was classified as unresponsive according to the rules stated in the protocol; however, the levels were so low in the beginning of PKU-003 and PKU-004 that a true measure was not possible c Double mutant; mutation is not completely classified Erlandsen et al 2004;Pey et al 2007;Scavelli et al 2005;Scriver and Waters 1999;Waters et al , 2000. Accordingly, it is relevant to know which among the several hundred PAH alleles (see www.pahdb.mcgill.ca) are associated with sapropterin responsiveness.…”

Linking genotypes database with locus-specific database and genotype–phenotype correlation in phenylketonuria

“…21 While multiple mechanisms for BH 4 responsiveness have been suggested, 22 in many cases BH 4 seems to act as a pharmacological chaperone assisting in protein folding, promoting enzyme stability and hindering degradation. 23,24 Response to BH 4 has also been shown to depend on initial blood Phe levels 21 and seems to require a certain minimal %PAH. 25 Additionally, genotype has been closely associated with BH 4 responsiveness type although not without inconsistencies.…”

Molecular Genetics and Genotype-Based Estimation of BH4-Responsiveness in Serbian PKU Patients: Spotlight on Phenotypic Implications of p.L48S

“…By binding to an unstable enzyme, BH4 protects it from degradation, prolongs its half-life and enables it to perform conversion of phenylalanine to tyrosine. (Erlandsen et al 2004;Pey et al 2004;Pérez et al 2005). The BH4-supplementation therapy (Kuvan) can be used to loosen or even replace burdensome dietary treatment of PKU patients (Levy et al 2007a;Trefz et al 2009b).…”