PKU MUTATION (D143G) ASSOCIATED WITH AN APPARENT HIGH RESIDUAL ENZYME-ACTIVITY - EXPRESSION OF A KINETIC VARIANT FORM OF PHENYLALANINE-HYDROXYLASE IN 3 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 kidn ey 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 sam e physicochemical properties as the wild-type PAH and was stable when expressed in eukaryotic cells, Enzyme activity studies of the D143G mu tant enzyme, produced in the three expression systems, revealed a kine tic variant form with reduced affinity for L-Phe (about 2.4-fold incre ase in the S-0.5 value) as well as reduced affinity for tetrahydrobiop terin (BH4) (about 2-fold increase in the apparent K-m). At standard a ssay conditions (1 mM L-Phe, 75 mu M BH4) the residual activity of the mutant enzyme was high and variable (52%, 33%, and 102%) when analyse d 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 f or the termination mutation G272X and the novel D143G mutation. Howeve r, when the D143G mutant enzyme was assayed at lower concentrations of L-Phe (100-300 mu M) and BH4 (10 mu M) the residual activities were c ompatible with severely reduced hydroxylation of L-Phe and the classic al PKU phenotype. (C) 1996 Wiley-Liss, Inc.