Mechanisms underlying responsiveness to tetrahydrobiopterin in mild phenylketonuria mutations

Abstract

A subtype of phenylalanine hydroxylase (PAH) deficiency that responds to cofactor (tetrahydrobiopterin, BH4) supplementation has been associated with phenylketonuria (PKU) mutations. The underlying molecular mechanism of this responsiveness is as yet unknown and requires a detailed in vitro expression analysis of the associated mutations. With this aim, we optimized the analysis of the kinetic and cofactor binding properties in recombinant human PAH and in seven mild PKU mutations, i.e., c.194T>C (p.I65T), c.204A>T (p.R68S), c.731C>T (p.P244L), c.782G>A (p.R261Q), c.926C>T (p.A309V), c.1162G>A (p.V388M), and c.1162G>A (p.Y414C) expressed in E. coli. For p.I65T, p.R68S, and p.R261Q, we could in addition study the equilibrium binding of BH4 to the tetrameric forms by isothermal titration calorimetry (ITC). All the mutations resulted in catalytic defects, and p.I65T, p.R68S, p.P244L, and most probably p.A309V, showed reduced binding affinity for BH4. The possible stabilizing effect of the cofactor was explored using a cell-free in vitro synthesis assay combined with pulse-chase methodology. BH4 prevents the degradation of the proteins of folding variants p.A309V, p.V388M, and p.Y414C, acting as a chemical chaperone. In addition, for wild-type PAH and all mild PKU mutants analyzed in this study, BH4 increases the PAH activity of the synthesized protein and protects from the rapid inactivation observed in vitro. Catalase and superoxide dismutase partially mimic this protection. All together, our results indicate that the response to BH4 substitution therapy by PKU mutations may have a multifactorial basis. Both effects of BH4 on PAH, i.e., the chemical chaperone effect preventing protein misfolding and the protection from inactivation, may be relevant mechanisms of the responsive phenotype.

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

“…Some mutants were obtained in the amounts required to perform isothermal titration calorimetry measurements and the thermodynamic binding parameters for BH 4 could be determined (Table 5, which is published as supporting information on the PNAS web site, and ref. 20). Defective BH 4 binding was confirmed by this technique for F39L and R68S, whereas I65T and A313T also showed a slightly decreased affinity.…”

“…HPLC and fluorescence detection of L-Tyr for nonactivated (non-L-Phe preincubated) and activated (L-Phe preincubated, 1 mM L-Phe) PAH enzymes were performed as described (18). Isothermal titration calorimetry (ITC) experiments were performed in a VP-ITC titration calorimeter (Microcal, Amherst, MA) at pH 7.0 with the glucose oxidase system (19) and 10-50 M PAH subunit with Ϸ0.5 mol ferrous ammonium sulfate͞subunit, as described (20). Additional information on the biophysical characterization is available in Table 3, which is which is published as supporting information on the PNAS web site.…”

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

“…40 The strong association between variants in the oligomerization domain and BH 4 responsiveness indicates that BH 4 most easily compensates oligomerization defects, which supports a role of BH 4 in oligomer assembly as suggested previously. 24 Overall, BH 4 responsiveness was best estimated by APV and %PAH and correctly predicted in 71.0% of all cases. In accordance with earlier findings, 25 BH 4 responsiveness required a minimal residual activity of about 20% in hemizygous genotypes, while the threshold was 15% in homozygous and compound heterozygous genotypes.…”

“…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).…”