Erlandsen Heidi, Pey Angel L, Gámez Alejandra, Pérez Belén, Desviat Lourdes R, Aguado Cristina, Koch Richard, Surendran Sankar, Tyring Stephen, Matalon Reuben, Scriver Charles R, Ugarte Magdalena, Martínez Aurora, Stevens Raymond C
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 2004 Nov 30;101(48):16903-8. doi: 10.1073/pnas.0407256101. Epub 2004 Nov 19.
Phenylketonuria patients harboring a subset of phenylalanine hydroxylase (PAH) mutations have recently shown normalization of blood phenylalanine levels upon oral administration of the PAH cofactor tetrahydrobiopterin [(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4)]. Several hypotheses have been put forward to explain BH4 responsiveness, but the molecular basis for the corrective effect(s) of BH4 has not been understood. We have investigated the biochemical, kinetic, and structural changes associated with BH4-responsive mutations (F39L, I65T, R68S, H170D, E178G, V190A, R261Q, A300S, L308F, A313T, A373T, V388M, E390G, P407S, and Y414C). The biochemical and kinetic characterization of the 15 mutants studied points toward a multifactorial basis for the BH4 responsiveness; the mutants show residual activity (>30% of WT) and display various kinetic defects, including increased Km (BH4) and reduced cooperativity of substrate binding, but no decoupling of cofactor (BH4) oxidation. For some, BH4 seems to function through stabilization and protection of the enzyme from inactivation and proteolytic degradation. In the crystal structures of a phenylketonuria mutant, A313T, minor changes were seen when compared with the WT PAH structures, consistent with the mild effects the mutant has upon activity of the enzyme both in vitro and in vivo. Truncations made in the A313T mutant PAH form revealed that the N and C termini of the enzyme influence active site binding. Of fundamental importance is the observation that BH4 appears to increase Phe catabolism if at least one of the two heterozygous mutations has any residual activity remaining.
携带苯丙氨酸羟化酶（PAH）部分突变的苯丙酮尿症患者最近显示，口服PAH辅因子四氢生物蝶呤[（6R）-L-赤藓糖-5,6,7,8-四氢生物蝶呤（BH4）]后血苯丙氨酸水平恢复正常。已经提出了几种假说来解释BH4反应性，但BH4纠正作用的分子基础尚未明确。我们研究了与BH4反应性突变（F39L、I65T、R68S、H170D、E178G、V190A、R261Q、A300S、L308F、A313T、A373T、V388M、E390G、P407S和Y414C）相关的生化、动力学和结构变化。对所研究的15个突变体的生化和动力学特征分析表明，BH4反应性存在多因素基础；这些突变体显示出残余活性（>野生型的30%），并表现出各种动力学缺陷，包括Km（BH4）增加和底物结合协同性降低，但辅因子（BH4）氧化未解偶联。对于一些突变体，BH4似乎通过稳定和保护酶使其免于失活和蛋白水解降解来发挥作用。在苯丙酮尿症突变体A313T的晶体结构中，与野生型PAH结构相比有微小变化，这与该突变体在体外和体内对酶活性的温和影响一致。对A313T突变体PAH形式进行截短后发现，酶的N端和C端影响活性位点结合。至关重要的是观察到，如果两个杂合突变中的至少一个仍有任何残余活性，BH4似乎会增加苯丙氨酸分解代谢。
