Manipulation of a cation-π sandwich reveals conformational flexibility in phenylalanine hydroxylase.
Manipulation of a cation-π sandwich reveals conformational flexibility in phenylalanine hydroxylase.
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DOI：
10.1016/j.biochi.2020.11.011
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发表时间：
2021-04
期刊：
影响因子：
3.9
通讯作者：
中科院分区：
文献类型：
--
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苯丙氨酸羟化酶（PAH）是一种变构酶，维持苯丙氨酸（Phe）低于神经毒性水平；它的失败导致苯丙酮尿症，这是一种先天性的氨基酸代谢错误。野生型（WT）多环芳烃在静息态（RS-PAH）和活化型（A-PAH）构象之间实现平衡，其平衡位置取决于变构苯醚结合。WT大鼠PAH （rPAH）的RS-PAH构象中含有一个涉及Phe80的阳离子-π三明治，这在a -PAH构象中是不存在的。制备了Phe80变异体F80A、F80D、F80L和F80R，并使用天然PAGE、大小排斥色谱、离子交换行为、内在蛋白荧光、酶动力学和有限蛋白水解等方法对其进行了评价，每一种都是[Phe]的函数。与WT - rpha一样，F80A和F80D也表现为Phe的变构激活，而F80L和F80R则表现为组成活性。所有变体的最大活性表明速率决定构象变化的缓解。WT - pah （- Phe）的有限蛋白水解揭示了埋在RS-PAH四聚体界面中的4-螺旋束的易裂解，反映了该四聚体的动态解离。这种切割在Phe80变异中没有观察到，在RS-PAH与a - pah交换过程中，所有的Phe80变异都表现出对连接体的蛋白水解过敏。通过添加Phe纠正超敏反应，使所有变体都变得像WT - pah，并达到A-PAH构象。因此，对Phe80的操纵扰乱了PAH采样的构象空间，增加了RS-PAH和A-PAH交换中通路上中间体的采样。Phe80变异体的行为与疾病相关的R68S相似，这表明pku相关的人类PAH变异体的蛋白水解易感性存在分子基础。
Phenylalanine hydroxylase (PAH) is an allosteric enzyme that maintains phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria, an inborn error of amino acid metabolism. Wild type (WT) PAH equilibrates among resting-state (RS-PAH) and activated (A-PAH) conformations, whose equilibrium position depends upon allosteric Phe binding. The RS-PAH conformation of WT rat PAH (rPAH) contains a cation-π sandwich involving Phe80, that cannot exist in the A-PAH conformation. Phe80 variants F80A, F80D, F80L, and F80R were prepared and evaluated using native PAGE, size exclusion chromatography, ion exchange behavior, intrinsic protein fluorescence, enzyme kinetics, and limited proteolysis, each as a function of [Phe]. Like WT rPAH, F80A and F80D show allosteric activation by Phe while F80L and F80R are constitutively active. Maximal activity of all variants suggests relief of a rate-determining conformational change. Limited proteolysis of WT rPAH (minus Phe) reveals facile cleavage within a 4-helix bundle that is buried in the RS-PAH tetramer interface, reflecting dynamic dissociation of that tetramer. This cleavage is not seen for the Phe80 variants, which all show proteolytic hypersensitivity in a linker that repositions during the RS-PAH to A-PAH interchange. Hypersensitivity is corrected by addition of Phe such that all variants become like WT rPAH and achieve the A-PAH conformation. Thus, manipulation of Phe80 perturbs the conformational space sampled by PAH, increasing sampling of on-pathway intermediates in the RS-PAH and A-PAH interchange. The behavior of the Phe80 variants mimics that of disease-associated R68S and suggests a molecular basis for proteolytic susceptibility in PKU-associated human PAH variants.
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影响因子：
8.8
作者：
Garbade, Sven F.;Shen, Nan;Blau, Nenad
通讯作者：
Blau, Nenad
DOI：
10.1073/pnas.1516967113
发表时间：
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发表时间：
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期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
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作者：
BUBLITZ, C
通讯作者：
BUBLITZ, C
影响因子：
4.8
作者：
Arturo, Emilia C.;Gupta, Kushol;Jaffe, Eileen K.
通讯作者：
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