Molecular mechanism for pterin-mediated inactivation of tyrosine hydroxylase: formation of insoluble aggregates of tyrosine hydroxylase.
Molecular mechanism for pterin-mediated inactivation of tyrosine hydroxylase: formation of insoluble aggregates of tyrosine hydroxylase.
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蝶呤介导的酪氨酸羟化酶失活的分子机制：酪氨酸羟化酶不溶性聚集体的形成。
DOI：
10.1093/jb/mvj073
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发表时间：
2006
影响因子：
2.7
通讯作者：
Ichinose,Hiroshi
中科院分区：
文献类型：
--
作者：
Urano,Fumi;Hayashi,Nobuhiro;Arisaka,Fumio;Kurita,Hideki;Murata,Shizuaki;Ichinose,Hiroshi
酪氨酸羟化酶（TH）是一种含铁酶，催化儿茶酚胺生物合成的第一步和限速步骤，需要四氢生物蝶呤（BH 4）作为辅因子。我们发现，预孵育的重组人TH与BH 4的结果在不可逆的失活的酶的浓度远低于theKm值对BH 4，尽管它的辅因子的作用，而氧化的生物蝶呤，它没有辅因子的活性，不影响酶的活性。我们表明，TH是失活的BH 4在竞争中与多巴胺的结合。顺序添加BH 4 TH的结果在荧光和CD光谱的强度逐渐降低，而不改变它们的整体配置文件。沉降速度分析表明，在BH 4的存在下，TH分子与彼此的关联，并使用凝胶渗透色谱法，浊度测量和透射电子显微镜的研究表明，形成无定形聚集体与大分子量的TH蛋白的关联后。这些结果表明，BH 4不仅作为辅助因子，而且还加速TH的聚集。我们提出了一种新的调节TH蛋白量的机制，并讨论了其生理意义。
Tyrosine hydroxylase (TH), an iron-containing enzyme, catalyzes the first and rate-limiting step of catecholamine biosynthesis, and requires tetrahydrobiopterin (BH4) as a cofactor. We found that preincubation of recombinant human TH with BH4 results in the irreversible inactivation of the enzyme at a concentration far less than theKmvalue toward BH4 in spite of its cofactor role, whereas oxidized biopterin, which has no cofactor activity, does not affect the enzyme activity. We show that TH is inactivated by BH4 in competition with the binding of dopamine. The sequential addition of BH4 to TH results in a gradual decrease in the intensity of the fluorescence and CD spectra without changing their overall profiles. Sedimentation velocity analysis demonstrated an association of TH molecules with each other in the presence of BH4, and studies using gel-permeation chromatography, turbidity measurements, and transmission electron microscopy demonstrated the formation of amorphous aggregates with large molecular weights following the association of the TH proteins. These results suggest that BH4 not only acts as a cofactor, but also accelerates the aggregation of TH. We propose a novel mechanism for regulating the amount of TH protein, and discuss its physiological significance.