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.

苯丙氨酸羟化酶 (PAH) 是一种变构酶，可将苯丙氨酸 (Phe) 维持在神经毒性水平以下；它的失败会导致苯丙酮尿症，这是氨基酸代谢的先天性错误。野生型 (WT) PAH 在静息态 (RS-PAH) 和激活 (A-PAH) 构象之间平衡，其平衡位置取决于变构 Phe 结合。WT 大鼠 PAH (rPAH) 的 RS-PAH 构象包含一个涉及 Phe80 的阳离子-π 三明治，它不能存在于 A-PAH 构象中。制备 Phe80 变体 F80A、F80D、F80L 和 F80R，并使用天然 PAGE、尺寸排阻色谱、离子交换行为、固有蛋白质荧光、酶动力学和有限蛋白水解进行评估，每个都是 [Phe] 的函数。与 WT rPAH 一样，F80A 和 F80D 显示 Phe 的变构激活，而 F80L 和 F80R 具有组成型活性。所有变体的最大活性表明决定速率的构象变化得到缓解。WT rPAH（减去 Phe）的有限蛋白水解揭示了埋藏在 RS-PAH 四聚体界面中的 4 螺旋束内的轻松切割，反映了该四聚体的动态解离。Phe80 变体没有看到这种切割，它们都显示在 RS-PAH 到 A-PAH 交换过程中重新定位的接头中的蛋白水解超敏反应。通过添加 Phe 纠正超敏反应，使所有变体变得像 WT rPAH 并实现 A-PAH 构象。因此，Phe80 的操作扰乱了 PAH 采样的构象空间，增加了 RS-PAH 和 A-PAH 交换中通路中间体的采样。