Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability
Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability
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DOI：
10.1016/j.ajhg.2008.05.013
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发表时间：
2008-07-01
影响因子：
9.8
通讯作者：
Muntau, Ania C.
中科院分区：
文献类型：
--
作者：
Gersting, Soren W.;Kemter, Kristina F.;Muntau, Ania C.
苯丙氨酸羟化酶（PAH）缺乏症的患者中有很大一部分受益于药理剂量的四氢生物蝶呤（BH 4），天然PAH辅因子。苯丙酮尿症（PKU）被假设为一种构象疾病，由于蛋白质不稳定而丧失功能，并且在BH 4治疗中观察到的酶功能的恢复可能通过纠正蛋白质错误折叠来传递。为了阐明PAH缺乏症功能障碍的分子基础，我们研究了在BH4反应性患者中鉴定的10种PAH基因突变对酶动力学、稳定性和蛋白质构象的影响（F55L、165S、H170Q、P275L、A300S、S310Y、P314S、R408W、Y414C、Y417H）。残余酶活性普遍较高，但变构在几乎所有情况下都受到干扰，并指出蛋白质构象的改变。这是证实了蛋白水解稳定性降低，受损的四聚体组装或聚集，增加疏水性，并加速热展开，特别是对监管结构域的影响，在大多数变体中观察到。三维建模揭示了功能相关的氨基酸网络的参与，可能会在整个蛋白质中传递错误折叠。我们的研究结果证实了PAH缺乏症是一种蛋白质错误折叠疾病，其中全局构象变化阻碍了生理酶功能所必需的分子运动。因此，PKU已经从导致严重神经损伤的遗传疾病的模型演变为具有功能丧失的可治疗的蛋白质折叠疾病的模型。
A significant share of patients with phenylalanine hydroxylase (PAH) deficiency benefits from pharmacological doses of tetrahydrobiopterin (BH4), the natural PAH cofactor. Phenylketonuria (PKU) is hypothesized to be a conformational disease, with loss of function due to protein destabilization, and the restoration of enzyme function that is observed in BH4 treatment might be transmitted by correction of protein misfolding. To elucidate the molecular basis of functional impairment in PAH deficiency, we investigated the impact of ten PAH gene mutations identified in patients with BH4-responsiveness on enzyme kinetics, stability, and conformation of the protein (F55L, 165S, H170Q, P275L, A300S, S310Y, P314S, R408W, Y414C, Y417H). Residual enzyme activity was generally high, but allostery was disturbed in almost all cases and pointed to altered protein conformation. This was confirmed by reduced proteolytic stability, impaired tetramer assembly or aggregation, increased hydrophobicity, and accelerated thermal unfolding-with particular impact on the regulatory domain-observed in most variants. Three-dimensional modeling revealed the involvement of functionally relevant amino acid networks that may communicate misfolding throughout the protein. Our results substantiate the view that PAH deficiency is a protein-misfolding disease in which global conformational changes hinder molecular motions essential for physiological enzyme function. Thus, PKU has evolved from a model of a genetic disease that leads to severe neurological impairment to a model of a treatable protein-folding disease with loss of function.