BIOSYNTHESIS OF CATECHOLAMINES

儿茶酚胺的生物合成

• 批准号: 3845191
• 负责人: S KAUFMAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3845191
• 关键词: Escherichia coli; chemical kinetics; dopamine; enzyme activity; enzyme mechanism; enzyme substrate; laboratory rat; molecular cloning; mutant; phosphorylation; posttranslational modifications; pteridines; recombinant DNA; synaptosomes; tyrosine 3 monooxygenase

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.

克隆大鼠嗜铬细胞瘤酪氨酸羟化酶，在大肠杆菌中表达， 随后纯化至均质。 纯的重组羟化酶 表现出与活化的天然酶相同的动力学行为。 然而，当用一种酶处理这种酶时， 儿茶酚胺类物质多巴胺 多巴胺被证明与 对重组酶的高亲和力，并引起显著变化， 酶的动力学和光谱特性。 结果 解释多巴胺缺乏时重组酶的激活 在大肠杆菌中，以及随之而来的缺乏多巴胺介导的下调， 羟化酶 此外，该数据确定多巴胺结合， 抑制作为一种必需真核生物翻译后修饰， 用于调节酪氨酸羟化酶活性。 几种缺失突变体 的酪氨酸羟化酶在大肠杆菌中表达并部分纯化， 进一步表征。 突变酶形式显示出不同的 催化活性水平和非常明显的动力学性质， 与缺失的大小和位置相关。 收效 有助于重新定义催化域的边界，并澄清 N-末端在指导底物特异性、辅因子结合中的作用 和产物抑制。 其他研究继续探索 酪氨酸羟化酶磷酸化和去磷酸化的机制 完整大鼠纹状体突触体。 早期的证据揭示了 酪氨酸羟化酶的去磷酸化，这是显着刺激 BH 4在原位。 目前的研究正试图确定具体的 BH 4的磷酸氨基酸靶位点。