MUTAGENESIS OF PYRIDOXAL PHOSPHATE DEPENDENT ENZYMES

磷酸吡哆醛依赖性酶的诱变

• 批准号: 2734529
• 负责人: JACK F KIRSCH
• 金额: $ 31.77万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734529
• 关键词: S adenosylmethionine; X ray crystallography; aspartate transaminase; carboxylate; cyclopropanes; enzyme activity; enzyme substrate; enzyme substrate complex; intermolecular interaction; malate dehydrogenase; nuclear magnetic resonance spectroscopy; oligonucleotides; pyridoxal phosphate; site directed mutagenesis; tyrosine transaminase

The principal objective of this grant is to explore the factors n enzyme design that allow discrimination between substrates. Two enzymes, aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase), both convert amino acids into 2- oxoacids. The Former is restrictive in substrate selectivity to the dicarboxylic amino acids, L-Asp and L-Glu, while the latter is permissive and accommodates, additionally, aromatic amino acids. Specificity is controlled by the side-chain of Arg292, which is rigid in AATase to keep out aromatic amino acids, but is mobile in TATase, and permits access of nearly all varieties of amino acids. We will use genetic engineering, in combination with kinetic techniques, to try to understand how the mobility of the side chain is restricted by the protein superstructure. Further genetic engineering experiments will be employed to reverse substrate charge specificity, and thus, convert AATase to an arginine transaminase. This will involve additional structure-based mutations of the ARG292 Asp mutation that we investigated nine years ago. Collaborative work with crystallographers in Switzerland is directed to structure elucidation of two new pyridoxal phosphate dependent enzymes, TATase discussed above, and ACC synthase, the key enzyme controlling ethylene production in plants. Ethylene is the plant ripening and senescence hormone. Additional goals are 1) to understand the details of the energetics of the ACC synthase mechanism by determining how the important intermediate on the reaction pathway partitions back to reactants or on to products; 2) to determine the transition state structure for the abstraction of the C alpha proton of S-adenosyl methionine in ACC synthase by Bronsted analysis; 3) to evaluate the significance of an unusual so- called, low barrier hydrogen bond in AATase by amino acid modification; 4) to develop a definitive technique to diagnose the existence of substrate channeling, whereby the product of the Nth enzyme in a metabolic pathway is delivered directly to the Nth+1 enzyme, for which it is a substrate, rather than being released first into solution.

这笔赠款的主要目的是探索影响因素 允许区分底物的酶设计。 二 酶、天冬氨酸转氨酶 (AATase) 和酪氨酸 转氨酶 (TATase)，均将氨基酸转化为 2- 含氧酸。 前者的底物选择性受到限制 二羧酸氨基酸，L-Asp 和 L-Glu，而后者是 允许并另外容纳芳香族氨基酸。 特异性由 Arg292 的侧链控制，即 AATase 呈刚性，可阻挡芳香族氨基酸，但可移动 TATase 中，并允许访问几乎所有种类的氨基 酸。 我们将利用基因工程，结合 动力学技术，试图了解 侧链受到蛋白质超结构的限制。 更远 基因工程实验将被用来逆转 底物电荷特异性，从而将 AATase 转化为 精氨酸转氨酶。 这将涉及额外的基于结构的 我们研究了九个 ARG292 Asp 突变 几年前。 与晶体学家的合作 瑞士致力于阐明两种新药物的结构 磷酸吡哆醛依赖性酶，上面讨论的 TATase， ACC合酶，控制乙烯的关键酶 工厂生产。 乙烯是植物成熟和 衰老激素。 其他目标是 1) 了解 ACC合酶机制的能量学细节 确定重要中间体对反应的影响 路径划分回到反应物或产物； 2）到 确定 C 抽象的过渡状态结构 ACC合酶中S-腺苷甲硫氨酸的α质子 布朗斯台德分析； 3）评估异常情况的重要性 AATase 中氨基酸形成的低势垒氢键 修改; 4）开发确定的技术来诊断 底物沟道的存在，其中第 N 个的产物 代谢途径中的酶直接传递至 Nth+1 酶，它是底物，而不是首先被释放 进入溶液。