PKU mutation (D143G) associated with an apparent high residual enzyme activity: expression of a kinetic variant form of phenylalanine hydroxylase in three different systems.

We have used three complementary in vitro systems to express the human phenylalanine hydroxylase (PAH) gene at high levels. Recombinant PAH was expressed in Escherichia coli (as a fusion protein), in human kidney cells and in a cell-free in vitro transcription-translation system. These systems were used to characterize a novel kinetic variant form (D143G) of the enzyme. The recombinant D143G mutant enzyme had the same physicochemical properties as the wild-type PAH and was stable when expressed in eukaryotic cells. Enzyme activity studies of the D143G mutant enzyme, produced in the three expression systems, revealed a kinetic variant form with reduced affinity for L-Phe (about 2.4-fold increase in the S0.5 value) as well as reduced affinity for tetrahydrobiopterin (BH4) (about 2-fold increase in the apparent Km). At standard assay conditions (1 mM L-Phe, t5 microM BH4) the residual activity of the mutant enzyme was high and variable (52%, 33%, and 102%) when analysed in the three different systems. The high residual activities of the mutant enzyme obtained at these conditions were not in agreement with the classical PKU phenotype found in a patient compound heterozygous for the termination mutation G272X and the novel D143G mutation. However, when the D143G mutant enzyme was assayed at lower concentrations of L-Phe (100-300 microM) and BH4 (10 microM) the residual activities were compatible with severely reduced hydroxylation of L-Phe and the classical PKU phenotype.