Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase
Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase
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DOI：
10.1016/j.abb.2010.10.009
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发表时间：
2011-01-15
影响因子：
3.9
通讯作者：
Fitzpatrick, Paul F.
中科院分区：
文献类型：
--
作者：
Li, Jun;Ilangovan, Udayar;Fitzpatrick, Paul F.
通过肝酶苯丙氨酸羟化酶将苯丙氨酸羟化为酪氨酸受苯丙氨酸水平调节。是否有一个独特的别构结合位点的活性位点外的苯丙氨酸一直不清楚。该酶包含一个延伸至Thr 117的N末端调节结构域。大鼠苯丙氨酸羟化酶调节结构域在大肠杆菌中表达。纯化的蛋白质在凝胶过滤柱上表现为二聚体。在苯丙氨酸的存在下，蛋白质从柱中较早洗脱，与氨基酸存在下的构象变化一致。在存在非活化氨基酸脯氨酸的情况下，未观察到洗脱变化。单独和在苯丙氨酸或脯氨酸存在下获得N-15标记的蛋白质的H-1-N-15 HSQC NMR谱。光谱中的峰的子集在苯丙氨酸的存在下表现出化学位移扰动，与苯丙氨酸在特定位点的结合一致。在脯氨酸的存在下，未观察到NMR谱的变化。这些结果表明，苯丙氨酸羟化酶的调节结构域可以结合苯丙氨酸，与氨基酸的变构位点的存在一致。(C)2010年爱思唯尔公司All rights reserved.
The hydroxylation of phenylalanine to tyrosine by the liver enzyme phenylalanine hydroxylase is regulated by the level of phenylalanine. Whether there is a distinct allosteric binding site for phenylalanine outside of the active site has been unclear. The enzyme contains an N-terminal regulatory domain that extends through Thr117. The regulatory domain of rat phenylalanine hydroxylase was expressed in Escherichia coli. The purified protein behaves as a dimer on a gel filtration column. In the presence of phenylalanine, the protein elutes earlier from the column, consistent with a conformational change in the presence of the amino acid. No change in elution is seen in the presence of the non-activating amino acid proline. H-1-N-15 HSQC NMR spectra were obtained of the N-15-labeled protein alone and in the presence of phenylalanine or proline. A subset of the peaks in the spectrum exhibits chemical shift perturbation in the presence of phenylalanine, consistent with binding of phenylalanine at a specific site. No change in the NMR spectrum is seen in the presence of proline. These results establish that the regulatory domain of phenylalanine hydroxylase can bind phenylalanine, consistent with the presence of an allosteric site for the amino acid. (C) 2010 Elsevier Inc. All rights reserved.