Nakashima A, Mori K, Suzuki T, Kurita H, Otani M, Nagatsu T, Ota A
Department of Physiology, School of Medicine, Fujita Health University, Toyoake, Aichi, Japan.
J Neurochem. 1999 May;72(5):2145-53. doi: 10.1046/j.1471-4159.1999.0722145.x.
Tyrosine hydroxylase (TH), which converts L-tyrosine to L-DOPA, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by feedback inhibition by catecholamine products including dopamine. To investigate the specific portion of the N-terminus of TH that determines the efficiency of dopamine inhibition, wild-type and N-terminal 35-, 38-, and 44-amino acid-deleted mutants (del-35, del-38, and del-44, respectively) of human TH type 1 were expressed as a maltose binding protein fusion in Escherichia coli and purified as a tetrameric form by affinity and size-exclusion chromatography. The fused-form wild-type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant nonfused form. Although maximum velocities of all N-terminus-deleted forms were about one-fourth of the wild-type value, there was no difference in Michaelis constants for L-tyrosine or (6R)-(L-erythro-1',2'-dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tetrahy dropteridine (6RBPH4) among the four enzymes. The iron contents incorporated into the three N-terminus-deleted mutants were significantly lower than that of wild type. However, there was no substantial difference in incorporated iron contents among the three mutants. The deletion of up to no less than 38 amino acid residues in the N-terminus made the enzyme more resistant to dopamine inhibition than the wild-type or del-35 TH form. Dopamine bound to the del-38 more than to the del-35 TH form. However, when incubation with dopamine was followed by further inhibition with the cofactor 6RBPH4 dopamine was expelled more readily from the del-38 than from the del-35 TH form. These observations suggest that the amino acid sequence Gly36-Arg37-Arg38 plays a key role in determining the competition between dopamine and 6RBPH4 and affects the efficiency of dopamine inhibition of the catalytic activity.
酪氨酸羟化酶（TH）可将L - 酪氨酸转化为L - 多巴，是儿茶酚胺生物合成中的限速酶；其活性受包括多巴胺在内的儿茶酚胺产物的反馈抑制调节。为了研究TH N端决定多巴胺抑制效率的特定部分，将人1型TH的野生型以及N端缺失35、38和44个氨基酸的突变体（分别为del - 35、del - 38和del - 44）作为麦芽糖结合蛋白融合体在大肠杆菌中表达，并通过亲和色谱和尺寸排阻色谱纯化得到四聚体形式。融合形式的野生型酶具有与先前报道的重组非融合形式几乎相同的比酶活性。尽管所有N端缺失形式的最大速度约为野生型值的四分之一，但这四种酶对L - 酪氨酸或（6R）-（L - 赤藓糖 - 1',2'-二羟基丙基）-2 - 氨基 - 4 - 羟基 - 5,6,7,8 - 四氢蝶呤（6RBPH4）的米氏常数没有差异。三个N端缺失突变体中掺入的铁含量明显低于野生型。然而，这三个突变体之间掺入的铁含量没有实质性差异。N端缺失多达38个氨基酸残基使该酶比野生型或del - 35 TH形式对多巴胺抑制更具抗性。多巴胺与del - 38的结合多于与del - 35 TH形式的结合。然而，当用多巴胺孵育后再用辅因子6RBPH4进一步抑制时，多巴胺从del - 38中比从del - 35 TH形式中更容易被排出。这些观察结果表明，氨基酸序列Gly36 - Arg37 - Arg38在决定多巴胺与6RBPH4之间的竞争中起关键作用，并影响多巴胺对催化活性抑制的效率。
