In vitro expression of 34 naturally occurring mutant variants of phenylalanine hydroxylase: Correlation with metabolic phenotypes and susceptibility toward protein aggregation

Phenylalanine hydroxylase (PAH) is a homotetrameric enzyme that catalyzes t he conversion of phenylalanine to tyrosine, the rate-limiting step of pheny lalanine disposal in humans. Primary dysfunction of PAH caused by mutations in the PAH gene results in hyperphenylalaninemia, which may impair cogniti ve development unless corrected by dietary restriction of phenylalanine. Th e mechanism(s) by which PAH missense mutations cause enzyme impairment has been studied in detail only in a small number of cases, but existing eviden ce points to a major role of enhanced proteolytic degradation due to aberra nt folding of mutant polypeptides. We have used two heterologous in vitro e xpression systems (a mammalian cell-free transcription-translation system a nd the pET system of Escherichia coli) to examine 34 mutations that have be en associated with PAH deficiency in the Danish population. These mutations represent a broad range of amino acid substitutions, functional enzyme dom ains, and metabolic phenotypes. In both systems, residual in vitro activiti es correlated broadly with metabolic phenotypes, however, with significant discrepancies. Analysis of E. coli extracts by nondenaturing polyacrylamide gel electrophoresis and storage experiments showed that (i) in general, mu tations in the N-terminal regulatory domain are associated with relatively stable proteins compared to most mutations in the central catalytic domain, and (ii) for mutations in the catalytic domain, high levels of protein agg regation do not always correspond with a severe phenotype. Our data support and extend previous evidence that PAH mutations exert their pathogenic eff ects by several distinct mechanisms that may operate individually or in con cert. (C) 2001 Academic Press.