Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, 11000, Belgrade, Serbia.
Protein J. 2009 Aug;28(6):294-9. doi: 10.1007/s10930-009-9194-z.
Phenylketonuria (PKU), the most frequent disorder of amino acid metabolism, is caused by mutations in human phenylalanine hydroxylase gene (PAH), leading to deficient enzyme activity. Previously reported but uncharacterized PAH gene mutation, p.S231F (c.692C > T), was detected in Serbian patients with classical PKU. We analyzed p.S231F PAH protein in prokaryotic (Escherichia coli) and eukaryotic expression system (hepatoma cells). In both systems the mutant enzyme was unstable. Residual enzyme activity in vitro was approximately 1%. Mutation p.S231F PAH was not activated by pre-incubation with phenylalanine substrate. We found no GroEL/GroES chaperone effect and slightly positive effect of the (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) on the stabilization of the protein structure. Our findings were in accordance with severe patients' phenotypes. In conclusion, p.S231F should be classified as a functionally null PAH gene mutation as it drastically reduces stability and activity of the PAH enzyme in vitro.
苯丙酮尿症（PKU）是最常见的氨基酸代谢紊乱，由人类苯丙氨酸羟化酶基因（PAH）突变引起，导致酶活性不足。先前在塞尔维亚的经典 PKU 患者中检测到了报道但未明确的 PAH 基因突变 p.S231F（c.692C > T）。我们在原核（大肠杆菌）和真核表达系统（肝癌细胞）中分析了 p.S231F PAH 蛋白。在这两种系统中，突变酶均不稳定。体外残余酶活性约为 1%。突变 p.S231F PAH 不能通过与苯丙氨酸底物预孵育而被激活。我们没有发现 GroEL/GroES 伴侣蛋白的效应，（6R）-L-erythro-5,6,7,8-四氢生物蝶呤（BH4）对蛋白质结构的稳定作用略有积极影响。我们的发现与严重患者的表型一致。总之，p.S231F 应被归类为功能缺失的 PAH 基因突变，因为它大大降低了 PAH 酶在体外的稳定性和活性。
