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 hy droxylase (PAH) gene has been characterized. An in vitro prokaryotic expres sion 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 fusi on proteins, were purified by immobilized metal affinity chromatography and recovered in high yields. The wild-type hPAH possessed a high specific act ivity and its kinetic properties were the same as those reported for the en zyme isolated from human liver and other recombinant wild-type hPAH enzymes . The recombinant V388M mutant form exhibited a reduced specific activity e quivalent to 30% of the wild-type hPAH enzyme when assayed using the synthe tic cofactor (6-methyltetrahydropterin). Lower values were obtained (23 and 19%) when the mutant enzyme was assayed with the natural cofactor ((6R)-te trahydrobiopterin) and different concentrations of L-phenylalanine. The enz yme kinetic studies of the V388M mutant protein revealed that this enzyme w as a kinetic variant form of hPAH with a reduced affinity for L-phenylalani ne and for the natural cofactor ((GR)-tetrahydrobiopterin), The residual ac tivities determined for the V388M form of hPAH were Compatible with the phe notype presented by the PKU patients harboring the V388M mutation in the PA H gene. (C) 2000 Academic Press.