PARTIAL CHARACTERIZATION AND 3-DIMENSIONAL STRUCTURAL LOCALIZATION OF8 MUTATIONS IN EXON-7 OF THE HUMAN PHENYLALANINE-HYDROXYLASE GENE ASSOCIATED WITH PHENYLKETONURIA
E. Bjorgo et al., PARTIAL CHARACTERIZATION AND 3-DIMENSIONAL STRUCTURAL LOCALIZATION OF8 MUTATIONS IN EXON-7 OF THE HUMAN PHENYLALANINE-HYDROXYLASE GENE ASSOCIATED WITH PHENYLKETONURIA, European journal of biochemistry, 257(1), 1998, pp. 1-10
The molecular basis for the metabolic defect in patients with phenylke
tonuria has been characterized for seven missense point mutations (R25
2G/Q, L255V/S, A259V/T and R270S) and a termination mutation (G272X) i
n an evolutionarily conserved motif of exon 7 in the catalytic domain
of the human phenylalanine hydroxylase (hPAH) gene. The mutations were
expressed in three heterologous in vitro systems, When expressed as f
usion proteins with maltose-binding protein in Escherichia coil five o
f the mutant proteins demonstrated a defect in the normal ability of h
PAH to fold and assemble as homotetramer/dimer, and they were mostly r
ecovered as inactive aggregated forms. Only for the R252Q and L255V mu
tants were catalytically active tetramer and dimer recovered and for R
252G some dimer, i.e. 20% (R252Q, tetramer), 44% (L255V, tetramer) and
4.4% (R252G, dimer) of the activity for the respective wild-type (wt)
forms. When expressed by a coupled ir? vitro transcription-translatio
n system, all the mutant enzymes were recovered as a mixture of non-ph
osphorylated and phosphorylated forms with a low homospecific activity
(i.e, maximum 11% of wt-hPAH for the L255V mutant). When transiently
expressed in human embryonic kidney (A293) cells a very low level of i
mmunoreactive PAH protein was recovered in spite of normal PAH mRNA le
vels. All these mutations resulted in variant hPAH proteins which reve
aled a defect in oligomerization, an increased sensitivity to limited
proteolysis in vitro, reduced cellular stability and a variable reduct
ion in their catalytic activity. All these effects seem to result from
structural perturbations of the monomer, and based on the crystal str
ucture of the catalytic domain of hPAH, an explanation is provided for
the impact of the mutations on the folding and oligomerization of the
monomers.