SITE DIRECTED MUTAGENESIS OF ASPARTATE AMINO TRANSFERASE

天冬氨酸氨基转移酶的定点诱变

• 批准号: 3288050
• 负责人: JACK F KIRSCH
• 金额: $ 21.84万
• 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/3288050
• 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.

本研究的目的和具体目标是 探讨天冬氨酸转氨酶的作用机制 （AAT酶）主要通过定点诱变， 半胱氨酸残基的化学加工引入到 活性部位采用相同的技术。 这些突变酶将 其物理特征在于圆二色性和微分 伯克利的扫描量热法; 合作和NMR合作动力学和机械 表征将继续在伯克利与快速和 传统的稳态动力学，荧光光谱，和 动力学同位素效应适合于每个突变体。 一个突变 已经被改造成阳离子氨基酸 特异性氨基转移酶将在这方面得到进一步发展， 通过合理选择的第二位点突变来增加 结合位点的负电荷密度，并通过选择 用质粒转化的精氨酸营养缺陷型 编码突变型AAT酶 有可能选择性地 压力会产生意想不到的第二位点突变体 的 这项工作与健康有关的方面来自一般作用， 磷酸吡哆醛依赖酶的氨基酸代谢 以及这些酶在先天性代谢缺陷中的作用， 高胱氨酸尿症、酪氨酸血症和缬氨酸血症。 生理 在代谢过程中相邻酶的结合具有重要意义 将通过扰动接触区域来研究路径， 化学修饰和定点诱变。 将通过以下方法观察络合物的稳定性： 差示扫描量热法;和化学性质 相关AAT酶的荧光，圆二色谱， 通过确定第一种酶的产物是否被引导 直接连接到第二个的活性位点。 更广泛的 研究的这一方面的影响适用于一般的 中间代谢的调节现象。