The V388M mutation results in a kinetic variant form of phenylalanine hydroxylase.

The molecular mechanism underlying the metabolic defect in phenylketonuria (PKU) patients carrying the V388M missense mutation of the phenylalanine hydroxylase (PAH) gene has been characterized. An in vitro prokaryotic expression system has been used to produce both the wild-type and the mutant form of the human PAH (hPAH) protein. The recombinant enzymes, obtained as fusion proteins, were purified by immobilized metal affinity chromatography and recovered in high yields. The wild-type hPAH possessed a high specific activity and its kinetic properties were the same as those reported for the enzyme isolated from human liver and other recombinant wild-type hPAH enzymes. The recombinant V388M mutant form exhibited a reduced specific activity equivalent to 30% of the wild-type hPAH enzyme when assayed using the synthetic cofactor (6-methyltetrahydropterin). Lower values were obtained (23 and 19%) when the mutant enzyme was assayed with the natural cofactor ((6R)-tetrahydrobiopterin) and different concentrations of l-phenylalanine. The enzyme kinetic studies of the V388M mutant protein revealed that this enzyme was a kinetic variant form of hPAH with a reduced affinity for l-phenylalanine and for the natural cofactor ((6R)-tetrahydrobiopterin). The residual activities determined for the V388M form of hPAH were compatible with the phenotype presented by the PKU patients harboring the V388M mutation in the PAH gene. Copyright 2000 Academic Press.