BIOSYNTHESIS OF CATECHOLAMINES

克隆了大鼠嗜铬细胞瘤酪氨酸羟基酶基因，并在大肠杆菌中表达， 并随后提纯至均一。纯净的重组羟基酶 表现出与激活的天然酶相同的动力学行为。 然而，当用一种药物处理酶时，这种激活被逆转。 儿茶酚胺产品中的多巴胺。多巴胺被证明与 对重组酶的高亲和力，并引起显著的变化 该酶的动力学和光谱性质。结果是 解释因缺乏多巴胺而激活重组酶的原因 在大肠杆菌中，以及由此导致的缺乏多巴胺介导的下调 羟基酶。此外，该日期确定了多巴胺结合和 抑制作为一种重要的真核细胞翻译后修饰 用于调节酪氨酸羟基酶的活性。几个缺失突变体 在大肠杆菌中表达了酪氨酸羟化酶，并对其进行了部分纯化 进一步的刻画。突变的酶形式表现出不同的 催化活性水平和非常独特的动力学特性， 与缺失的大小和位置相关。结果是 帮助重新定义了催化域的边界并澄清了 N末端在引导底物特异性、辅因子结合中的作用 和产物抑制。其他研究一直在继续探索 酪氨酸羟基酶的磷酸化和去磷酸化机制 完整的大鼠纹状体突触体。更早的证据揭示了一条 酪氨酸羟基酶的去磷酸化作用 BH4原位。目前的研究正试图确定特定的 BH4的磷酸氨基酸靶位。

Rat pheochromochytoma tyrosine hydroxylase was cloned, expressed in E.coli, and subsequently purified to homogeneity. The pure recombinant hydroxylase exhibited the same kinetic behavior as that of the activated native enzyme. This activation was reversed, however, when the enzyme was treated with one of the catecholamine products, dopamine. Dopamine was shown to bind with high-affinity to the recombinant enzyme and to cause a significant change i the kinetic as well as spectral properties of the enzyme. The results explain the activation of the recombinant enzyme by the absence of dopamine in E.coli, and the consequent lack of dopamine-mediated down regulation of the hydroxylase. Furthermore, the date identify dopamine binding and inhibition as an essential eukaryotic post-translational modification that serves to regulate tyrosine hydroxylase activity. Several deletion mutants of tyrosine hydroxylase were expressed in E.coli and partially purified for further characterization. The mutant enzyme forms displayed different levels of catalytic activity and very distinct kinetic properties, which were correlated to the size and location of the deletion. The results have helped to redefine the boundaries of the catalytic domain and to clarify th role of the N-terminus in directing substrate specificy, cofactor binding and product inhibition. Other studies have continued to explore the mechanism of tyrosine hydroxylase phosphorylation and dephosphorylation in intact rat striatal synaptosomes. Earlier evidence unveiled a pathway of dephosphorylation for tyrosine hydroxylase which was markedly stimulated by BH4 in situ. Current studies are attempting to identy the specific phosphoamino acid target sites for BH4.