The regulatory domain of human tryptophan hydroxylase 1 forms a stable dimer.
The regulatory domain of human tryptophan hydroxylase 1 forms a stable dimer.
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DOI：
10.1016/j.bbrc.2016.05.144
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发表时间：
2016-08-05
影响因子：
3.1
通讯作者：
Fitzpatrick PF
中科院分区：
文献类型：
--
作者：
Zhang S;Hinck CS;Fitzpatrick PF
三种真核芳香族氨基酸羟化酶苯丙氨酸羟化酶、酪氨酸羟化酶和色氨酸羟化酶具有基本上相同的催化结构域和离散的调节结构域。当苯丙氨酸结合到变构位点时，苯丙氨酸羟化酶的调节结构域形成ACT结构域二聚体。相反，酪氨酸羟化酶的调节结构域形成不结合氨基酸底物的稳定ACT二聚体。表达并纯化了人色氨酸羟化酶亚型1的调节结构域;需要对Cys 64进行诱变以防止形成二硫键连接的二聚体。所得蛋白质在凝胶过滤和分析超离心时表现为二聚体。蛋白质的sw值在2.7-35 µM（苯丙氨酸羟化酶的调节结构域形成单体和二聚体的浓度范围）内未发生变化，这与色氨酸羟化酶1的调节结构域形成稳定的二聚体（不经历单体-二聚体平衡）一致。添加苯丙氨酸（酶的良好底物）对sw值没有影响，这与氨基酸底物没有变构位点一致。
The three eukaryotic aromatic amino acid hydroxylases phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase have essentially identical catalytic domains and discrete regulatory domains. The regulatory domains of phenylalanine hydroxylase form ACT domain dimers when phenylalanine is bound to an allosteric site. In contrast the regulatory domains of tyrosine hydroxylase form a stable ACT dimer that does not bind the amino acid substrate. The regulatory domain of isoform 1 of human tryptophan hydroxylase was expressed and purified; mutagenesis of Cys64 was required to prevent formation of disulfide-linked dimers. The resulting protein behaved as a dimer upon gel filtration and in analytical ultracentrifugation. The sw value of the protein was unchanged from 2.7–35 µM, a concentration range over which the regulatory domain of phenylalanine hydroxylase forms both monomers and dimers, consistent with the regulatory domain of tryptophan hydroxylase 1 forming a stable dimer stable that does not undergo a monomer-dimer equilibrium. Addition of phenylalanine, a good substrate for the enzyme, had no effect on the sw value, consistent with there being no allosteric site for the amino acid substrate.