Molecular mechanisms of PAH function in response to phenylalanine and tetrahydrobiopterin binding

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.