SITE DIRECTED MUTAGENESIS OF ASPARTATE AMINO TRANSFERASE

天冬氨酸氨基转移酶的定点诱变

• 批准号: 3288051
• 负责人: JACK F KIRSCH
• 金额: $ 22.71万
• 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 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288051
• 关键词: Escherichia coli; X ray crystallography; aspartate transaminase; bacterial genetics; calorimetry; chemical binding; cysteine; enzyme mechanism; enzyme structure; enzyme substrate; genetic manipulation; glutamine; histidine; malate dehydrogenase; nuclear magnetic resonance spectroscopy; nucleic acid sequence; point mutation; protein engineering; protein sequence; site directed mutagenesis

The objectives and specific aims of this research are to investigate the mechanism of action of aspartate aminotransferase (AATase) primarily through site-directed mutagenesis-and by chemical elaboration of cysteine residues introduced into the active site by the same technique. These mutant enzymes will be characterized physically by circular dichroism and differential scanning calorimetry in Berkeley; by crystallography in collaboration and by NMR in collaboration Kinetic and mechanistic characterization will be continued in Berkeley with rapid and conventional steady-state kinetics, fluorescence spectroscopy, and kinetic isotope effects as appropriate to each mutant. One mutant that has already been engineered to be a cationic amino acid- specific aminotransferase will be further developed in this direction by a rationally selected second-site mutation to increase the negative-charge density at the binding site, and by selecting arginine auxotrophs which have been transformed with the plasmid coding for the mutant AATase. It is possible that selective pressure will produce unanticipated second-site mutants. The health-related aspects of this work derive from the general role of pyridoxal phosphate-dependent enzymes in amino acid metabolism and of these enzymes in inborn errors of metabolism, such as homocystinuria, tyrosinemia, and valinemia. The physiological significant of the association of adjacent enzymes in metabolic pathways will be investigated by perturbing the contact areas by chemical modification and by site-directed mutagenesis. Observations on the stability of the complexes will be made by differential scanning calorimetry; and on the chemical properties of the associated AATase by fluorescence, circular dichroism, and by determining whether the product of the first enzyme is channeled directly to the active site of the second. The broader implications of the aspect of the research apply to the general phenomenon of the regulation of intermediary metabolism.

这项研究的目标和具体目的是 研究天冬氨酸氨基转移酶的作用机理 （AATASE）主要通过定点诱变和 引入的半胱氨酸残基的化学阐述 通过相同技术的活动站点。 这些突变酶将是 通过圆形二科和差异的身体特征 在伯克利扫描量热法；通过晶体学IN 合作以及NMR协作动力学和机械 伯克利将继续以快速和 常规的稳态动力学，荧光光谱和 动力学同位素效应适合每个突变体。 一个突变体 已经设计为阳离子氨基酸 - 特定的氨基转移酶将进一步开发 通过合理选择的第二站点突​​变的方向增加 结合位点的负电荷密度，并选择 精氨酸的合子已与质粒转化 编码突变体AATASE。 有可能有选择性 压力将产生意外的第二站点突​​变体。 这 这项工作的与健康相关的方面源于一般角色 氨基酸代谢中吡啶还毒素依赖性酶的 以及这些酶在天生的新陈代谢错误中，例如 同胞肠尿症，酪氨酸血症和valinemia。 生理 代谢中相邻酶的关联很大 将通过扰动联系区域来调查途径 化学修饰和通过位置定向诱变。 关于复合物稳定性的观察将由 差扫描量热法；并在化学特性上 通过荧光，圆形二色性和 通过确定是否引导第一种酶的乘积 直接进入第二个的活动位置。 更广泛的 研究方面的含义适用于一般 中间代谢调节的现象。