MUTAGENESIS OF PYRIDOXAL PHOSPHATE-DEPENDENT ENZYMES

本研究的目的和具体目标是：1）重新设计 天冬氨酸氨基转移酶的催化特异性， 使二羧酸氨基酸天冬氨酸和谷氨酸转氨， 酪氨酸氨基转移酶具有更宽的氨基酸 的特异性天冬氨酸氨基转移酶的定点突变将 基于两种同源结构的计算机建模。2)在 体外翻译将用于引入非天然氨基酸， 特别是天冬氨酸转氨酶的关键位点， 在机制和底物特异性中插入更微妙的变化 比从标准的十九种氨基酸中得到的还要多 可通过定点诱变技术获得。3)的缺失 活性位点赖氨酸侧链（K258 A）将在tryrosme中制备 转氨酶这种酶催化取代的 苯基甘氨酸。因此，将有可能首次研究 同一种酶的经典Bronsted关系和Hammett关系 催化反应。结果应提供有关反应的详细信息 坐标表面以及过渡态结构。4)网站 定向诱变将用于探索Cys 191 保留在天冬氨酸转氨酶同工酶中，因为 陷入了一个进化的井，从那里没有单一的碱基变化， 逃跑5)我们将使用荧光光谱和其他物理 试图发现缓慢的物理基础， 动力学活性，（t1/2=10分钟）引入构象变化 通过突变D222 A，和7）氨基环丙烷的活性位点 羧酸合酶（ACC合酶）在植物中几乎100%保守 与天冬氨酸转氨酶比较。前一种酶， 对于植物成熟激素乙烯的生物合成很重要， 将在E.大肠杆菌或酵母菌， 活性位点氨基酸突变。突变将基于我们的 目前对天冬氨酸中相应取代的理解 转氨酶

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.