title: Characterization of phenylketonuria missense substitutions, distant from the phenylalanine hydroxylase active site, illustrates a paradigm for mechanism and potential modulation of phenotype.
abstract: Missense mutations account for 48% of all reported human disease-causing alleles. Since few are predicted to ablate directly an enzyme's catalytic site or other functionally important amino acid residues, how do most missense mutations cause loss of function and lead to disease? The classic monogenic phenotype hyperphenylalaninemia (HPA), manifesting notably as phenylketonuria (PKU), where missense mutations in the PAH gene compose 60% of the alleles impairing phenylalanine hydroxylase (PAH) function, allows us to examine this question. Here we characterize four PKU-associated PAH mutations (F39L, K42I, L48S, I65T), each changing an amino acid distant from the enzyme active site. Using three complementary in vitro protein expression systems, and 3D-structural localization, we demonstrate a common mechanism. PAH protein folding is affected, causing altered oligomerization and accelerated proteolytic degradation, leading to reduced cellular levels of this cytosolic protein. Enzyme specific activity and kinetic properties are not adversely affected, implying that the only way these mutations reduce enzyme activity within cells in vivo is by producing structural changes which provoke the cell to destroy the aberrant protein. The F39L, L48S, and I65T PAH mutations were selected because each is associated with a spectrum of in vivo HPA among patients. Our in vitro data suggest that interindividual differences in cellular handling of the mutant, but active, PAH proteins will contribute to the observed variability of phenotypic severity. PKU thus supports a newly emerging paradigm both for mechanism whereby missense mutations cause genetic disease and for potential modulation of a disease phenotype.
authors.affiliation: deBelle Laboratory for Biochemical Genetics, Montreal Children's Hospital, Montreal, H3H 1P3, Canada. pjwaters@www.debelle
authors.forename: P J
authors.initials: PJ
authors.lastname: Waters
authors.forename: M A
authors.initials: MA
authors.lastname: Parniak
authors.forename: B R
authors.initials: BR
authors.lastname: Akerman
authors.forename: C R
authors.initials: CR
authors.lastname: Scriver
mesh_terms: D018528:ATP-Binding Cassette Transporters; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000235:genetics; D020134:Catalytic Domain; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D006801:Humans; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D020125:Mutation, Missense; D010641:Phenotype; D010651:Phenylalanine Hydroxylase / Q000235:genetics* / Q000378:metabolism; D010661:Phenylketonurias / Q000235:genetics*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D010651:Phenylalanine Hydroxylase