Phenylalanine hydroxylase. Evidence that the enzyme from human liver might not be a phosphoprotein.

Phenylalanine hydroxylase from fresh human liver was purified to homogeneity with a 60% yield by a three steps procedure involving hydrophobic chromatography on Phenyl-Sepharose, ion exchange chromatography on DEAE-Cellulose and High Performance gel permeation chromatography. The purified native enzyme had an estimated molecular weight of 165,000. It gave a single band on Sodium Dodecylsulfate polyacrylamide gel electrophoresis under an estimated molecular weight of 55,000. Neither the purified human enzyme, nor that present in a liver extract could be activated under phosphorylating conditions. Moreover, the purified human liver phenylalanine hydroxylase was found to be devoid of protein-bound phosphate and no phosphate could be incorporated from [32P]-ATP in the presence of cyclic-AMP - dependent protein kinase. These data suggest that phenylalanine hydroxylase from human liver, unlike that of rat liver, might not be a phosphoprotein.

The mutant genotype is the main determinant of the metabolic phenotype in phenylalanine hydroxylase deficiency.

Phenylketonuria and mild hyperphenylalaninemias are allelic disorders caused by mutations in the phenylalanine hydroxylase (PAH) gene. Following identification of the disease-causing mutation in 11 PAH-deficient patients, we tested the activity of the mutant gene products in an eukaryotic expression system. Two mutations markedly reduced PAH activity (A259V and L333F), one mutation mildly altered the enzyme activity (E390G), while the majority of mutant genotypes reduced the in vitro expression of PAH activity to 15-30% of controls. Comparing the predicted residual activity derived from expression studies to the clinical phenotypes of our PAH-deficient patients, we found that homozygosity for the L333F and E390G mutations resulted in severe and mild PAH deficiencies, respectively, both in vivo and in vitro, while compound heterozygosity (L333F/E390G) resulted in an intermediate dietary tolerance. Similarly, in vitro expression studies largely predicted dietary tolerance in compound heterozygotes for the A259V/IVS12nt1 (typical PKU), A259V/A403V, G218V/I65T, and G218V/R158Q mutations (mild variants). Taken together, these results support the view that expression studies are useful in predicting residual enzyme activity and that the mutant genotype at the PAH locus is the major determinant of metabolic phenotype in hyperphenylalaninemias.

Clinical and molecular heterogeneity of phenylalanine hydroxylase deficiencies in France.

RFLPs of 68 normal and 74 mutant alleles at the phenylalanine hydroxylase (PAH) locus were determined in 37 French kindreds. A total of 23 haplotypes, including 18 normal and 16 mutant alleles, were observed. Two-thirds of all mutant alleles were confined within only four haplotypes, while the last third was accounted for by 12 haplotypes, including eight haplotypes absent from Caucasian pedigrees reported thus far. Several mutant haplotypes were present in typical phenylketonuria only, others were present in variants only, and some were present in both. In addition, a particular mutant haplotype (haplotype 2) was found to harbor different mutations in our series, resulting in either typical phenylketonuria or in mild hyperphenylalaninemias. The diploid combination of so many mutant haplotypes in PAH-deficient patients and of compound heterozygosity at the PAH locus in southern Europe might account for the broad spectrum of individual phenotypes observed in France.