MUTAGENESIS OF PYRIDOXAL PHOSPHATE-DEPENDENT ENZYMES

磷酸吡哆醛依赖性酶的诱变

• 批准号: 2177879
• 负责人: JACK F KIRSCH
• 金额: $ 30.07万
• 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 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177879
• 关键词: MUTAGENESIS; PYRIDOXAL; PHOSPHATE; DEPENDENT; ENZYMES

The objectives and specific aims of this research are: 1) To redesign the catalytic specificity of aspartate aminotransferase, an enzyme which transaminates the dicarboxylic amino acids aspartate and glutamate, to that of tyrosine aminotransferase which has a much broader amino acid specificity. Site directed mutagenesis of aspartate aminotransferase will be based on computer modeling of the two homologous structures. 2) In vitro translation will be used to introduce unnatural amino acids at particularly crucial loci in aspartate aminotransferase in order to interject more subtle variations in mechanism and substrate specificity than can be obtained from the standard set of nineteen amino acids obtainable by site directed mutagenesis technology. 3) A deletion of the active site lysine side chain (K258A) will be made in tryrosme aminotransferase. This enzyme catalyzes the transamination of substituted phenyl glycines. It will therefore be possible for the first time to study both classical Bronsted and Hammett relations for the same enzyme catalyzed reactions. The results should provide details about the reaction coordinate surface as well as of the transition state structure. 4) Site directed mutagenesis will be used to explore the hypothesis that Cys191 has been retained in aspartate aminotransferase isozymes, because it was caught in an evolutionary well from which there is no single base change escape. 5) We will use fluorescence spectroscopy and other physical methods to attempt to discover the physical basis for the slow, kinetically competent, (t1/2=10 minutes) conformational change introduced by the mutation D222A, and 7) The active site of amino cydopropane carboxylate synthase (ACC synthase) is virtually 100% conserved in comparison to aspartate aminotransferase. The former enzyme, which is important for the biosynthesis of the plant ripening hormone ethylene, will be heterologously expressed in E. coli or yeast, and the putative active site amino acids mutated. The mutations will be based on our present understanding of the corresponding substitutions in aspartate aminotransferase.

本研究的目的和具体目的是：1)重新设计 天冬氨酸氨基转移酶的催化专一性 将天冬氨酸和谷氨酸的二羧酸转氨酶 酪氨酸氨基转移酶具有更广泛的氨基酸 专一性。天冬氨酸氨基转移酶Will的定点突变 基于两个同源结构的计算机建模。2)在 体外翻译将用于将非天然氨基酸引入 天冬氨酸氨基转移酶中特别关键的基因座 插入机制和底物专一性的更微妙的变化 比从19种氨基酸的标准集合中得到的更多 可通过定点突变技术获得。3)删除 活性中心赖氨酸侧链(K258A)将在trirosme中制造 转氨酶。这种酶催化取代基团的转氨基 苯甘氨酸。因此，将有可能第一次研究 同一种酶的经典Bronsted和Hammett关系 催化反应。结果应该提供关于反应的细节 以及过渡态结构的坐标曲面。4)站点 定向突变将被用于探索Cys191的假设 保留在天冬氨酸氨基转移酶同工酶中，因为它是 被困在进化井中，没有任何单一的碱基变化 逃走。5)我们将使用荧光光谱和其他物理方法 试图发现慢性病的物理基础， 运动能力强，(T1/2=10分钟)引入构象变化 突变D222a，以及7)氨基环丙烷的活性部位 羧酸合成酶(ACC合成酶)几乎是100%保守的 与天冬氨酸氨基转移酶的比较。前一种酶，即 对植物成熟激素乙烯的生物合成很重要， 将在大肠杆菌或酵母中异源表达，并推定 活性部位氨基酸发生突变。突变将基于我们的 目前对天冬氨酸中相应取代基的理解 转氨酶。