MUTAGENESIS OF PYRIDOXAL PHOSPHATE-DEPENDENT ENZYMES

磷酸吡哆醛依赖性酶的诱变

• 批准号: 2177878
• 负责人: JACK F KIRSCH
• 金额: $ 30.01万
• 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/2177878
• 关键词: Escherichia coli; S adenosylmethionine; X ray crystallography; active sites; aminoacid analog; aspartate transaminase; carbon sulfur lyase; computer simulation; cysteine; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; fluorescence spectrometry; gene deletion mutation; genetic translation; isozymes; protein sequence; pyridoxal phosphate; site directed mutagenesis; tyrosine transaminase

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.

这项研究的目标和具体目的是：1）重新设计 天冬氨酸氨基转移酶的催化特异性，一种酶，该酶 将二羧酸氨基酸天冬氨酸和谷氨酸转移到 酪氨酸氨基转移酶的氨基酸的氨基转移酶 特异性。地点定向天冬氨酸氨基转移酶的诱变将 基于两个同源结构的计算机建模。 2）in 体外翻译将用于引入不自然的氨基酸 特别是在天冬氨酸氨基转移酶中的关键基因座 插入机制和底物特异性方面的更微妙的变化 比从标准的19个氨基酸集获得 可以通过站点定向诱变技术获得。 3）删除 活跃部位赖氨酸侧链（K258A）将在Tryrosme中进行 氨基转移酶。这种酶催化取代 苯基甘氨酸。因此，第一次有可能学习 相同酶的经典布朗斯特和哈米特关系 催化反应。结果应提供有关反应的详细信息 协调表面以及过渡状态结构。 4）站点 定向诱变将用于探索Cys191的假设 已保留在天冬氨酸氨基转移酶同工酶中，因为它是 陷入了没有单一基础变化的进化井中 逃脱。 5）我们将使用荧光光谱和其他物理 试图发现慢速的物理基础的方法 动力学胜任（T1/2 = 10分钟）引入了构象变化 通过突变D222a和7）氨基葡萄丙烷的活性位点 羧酸盐合酶（ACC合酶）实际上是100％保守的 与天冬氨酸氨基转移酶进行比较。以前的酶， 对于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 活性位点氨基酸突变。突变将基于我们 目前对天冬氨酸中相应替代的理解 氨基转移酶。