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Danecka, Marta Kinga (2016): Molecular mechanisms of PAH function in response to phenylalanine and tetrahydrobiopterin binding: implications for clinical management. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism (IEM) caused by mutations in the phenylalanine hydroxylase (PAH) gene. The molecular mechanism underlying deficiency of the PAH protein is, in most of the cases, loss of function due to protein misfolding. PAH mutations induce disturbed oligomerisation, decreased stability and accelerated degradation of hepatic PAH, a key enzyme in phenylalanine metabolism. Since the development of a phenylalanine-restricted diet in the 1950ies, PKU is a prototype for treatable inherited diseases. About 60 years later, the natural PAH cofactor tetrahydrobiopterin (BH4) was shown to act as a pharmacological chaperone stabilising the misfolded PAH protein. In consequence, BH4 (KUVAN®) was introduced to the pharmaceutical market as an alternative treatment for BH4-responsive PAH deficiency. Therefore, PKU is also regarded as a prototype for a pharmacologically treatable protein misfolding disease. Despite the progress in PKU therapy, knowledge on the molecular basis of PKU and the BH4 mode of action was still incomplete. Biochemical and biophysical characterisation of purified variant PAH proteins, which were derived from patient’s mutations, aimed at a better understanding of the molecular mechanisms of PAH loss of function. We showed that local side-chain replacements induce global conformational changes with negative impact on molecular motions that are essential for physiological enzyme function. The development of a continuous real-time fluorescence-based assay of PAH activity allowed for robust analysis of steady state kinetics and allosteric behaviour of recombinantly expressed PAH proteins. We identified positive cooperativity of the PAH enzyme towards BH4, where cooperativity does not rely on the presence of phenylalanine but is determined by activating conformational rearrangements. In vivo investigations on the mode-of-action of BH4 revealed differences in pharmacodynamics but not in pharmacokinetics between different strains of PAH-deficient mice (wild-type, Pahenu1/1 and Pahenu1/2). These observations pointed to a significant impact of the genotype on responsiveness to BH4. The available database information on PAH function associated with PAH mutations was based on non-standardised enzyme activity assays performed in different cellular systems and under different conditions usually focusing on single PAH mutations. These inconsistent data on PAH enzyme activity hindered robust prediction of the patient’s phenotype. Furthermore, assays on single PAH mutations do not reflect the high allelic and phenotypic heterogeneity of PKU with 89 % of patients being compound heterozygotes. In addition, the knowledge on enzyme function and regulation in the therapeutic and pathologic metabolic context was still scarce. In order to get more insight into the interplay of the PAH genotype, the phenylalanine concentration and BH4 treatment, we performed functional analyses of both, single, purified PAH variants as well as PAH full genotypes in the physiological, pathological and therapeutic context. The analysis of PAH activity as a function of phenylalanine and BH4 concentrations enabled determination of the optimal working ranges of the enzyme and visualisation of differences in the regulation of PAH activity by BH4 and phenylalanine depending on the underlying genotype. Moreover, these PAH activity landscapes allowed for setting rules for dietary regimens and pharmacological treatment based on the genotype of the patient. Taken together, precise knowledge on the mechanism of the misfolding-induced loss of function in PAH deficiency enabled a better understanding of the molecular mode of action of pharmacological rescue of enzyme function by BH4. We implemented the combination of genotype-specific functional analyses together with biochemical, clinical and therapeutic data of individual patients as a powerful tool for phenotype prediction and paved the way for personalised medicine strategies in phenylketonuria.