Characterization of phenylketonuria missense substitutions, distant from the phenylalanine hydroxylase active site, illustrates a paradigm for mechanism and potential modulation of phenotype

Missense mutations account for 48% of all reported human disease-causing al leles. Since few are predicted to ablate directly an enzyme's catalytic sit e or other functionally important amino acid residues, how do most missense mutations cause loss of function and lead to disease? The classic monogeni c phenotype hyperphenylalaninemia (HPA), manifesting notably as phenylketon uria (PKU), where missense mutations in the PAH gene compose 60% of the all eles impairing phenylalanine hydroxylase (PAH) function, allows us to exami ne this question. Here we characterize four PW-associated PAH mutations (F3 9L, 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 pro tein folding is affected, causing altered oligomerization and accelerated p roteolytic degradation, leading to reduced cellular levels of this cytosoli c protein. Enzyme specific activity and kinetic properties are not adversel y affected, implying that the only way these mutations reduce enzyme activi ty within cells in vivo is by producing structural changes which provoke th e cell to destroy the aberrant protein. The F39L, L48S, and I65T PAH mutati ons 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 contribu te 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. (C) 2 000 Academic Press.