SITE-SPECIFIC MUTAGENESIS OF ISOMERASES

异构酶的位点特异性诱变

• 批准号: 3281221
• 负责人: GREGORY A PETSKO
• 金额: $ 17.84万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-16 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3281221
• 关键词: Streptomyces; X ray crystallography; active sites; chemical binding; chemical kinetics; conformation; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; inborn carbohydrate metabolism disorder; isomerase; mutant; point mutation; protein engineering; protein sequence; site directed mutagenesis; thermodynamics; thermostability; triose phosphate isomerase; xylose

The goal of this project is to use a combination of site-directed mutagenesis and X-ray crystallography to understand the structural basis of the catalytic power of yeast triosephosphate isomerase (TIM) and to unravel the mechanism of action of xylose isomerase (also called glucose isomerase and abbreviated XyI). Triosephosphate isomerase is the central enzyme in the glycolytic pathway and is extremely efficient: it operates at the diffusion-controlled limit. Xylose isomerase is the most widely-used industrial enzyme, is of paramount importance to the food industry, and is very inefficient. It operates over 100,000 times slower than TIM. Yet the two enzymes catalyse the same chemical transformation, the interconversion of an aldehyde and a ketone. Understanding the catalytic efficiency of TIM is important because there is a terrible human TIM deficiency disease. It is an inborn error of metabolism, inherited in an automosomal recessive fashion, and it leads to acute hemolytic anemia, severe neurological disfunction, increased susceptibility to infection, and propensity for sudden cardiac death. The lesion in several patients appears to be the mutation of a single amino acid, Glu 104, to an aspartic acid. One of the specific aims of this proposal is to duplicate this human TIM mutant in yeast TIM, characterize the kinetics and stability of the altered protein, and determine its three-dimensional structure. Comparison of this structure with that of the wild-type enzyme may lead to an under- standing of how this substitution leads to a deadly inherited metabolic disease. Other specific goals focus on understanding the roles of specific amino acids in the catalytic activity of both enzymes. The residues in question will be altered by sitedirected mutagenesis and the properties of the mutant proteins will be determined. One advantage of the TIM system is that the complete free-energy profile can be determined for the reaction catalysed by any interesting mutant, so the exact microsteps affected by the mutation can be discovered. Crystal structures will be obtained for every mutant in complex with a tight-binding inhibitor that is an analog of the intermediate in the reaction. For XyI, every mutant will be characterized in terms of its effect on the two separate stages of the reaction: catalytic opening of the sugar ring and isomerization. Every mutant will also be examined crystallographically, in complex with the actual substrate glucose.

这个项目的目标是使用站点定向的组合 用诱变和X射线结晶学来了解其结构基础 酵母磷酸丙糖异构酶(TIM)的催化能力 解开木糖异构酶(又称葡萄糖)的作用机制 异构酶和缩写XyI)。磷酸丙糖异构酶是中枢 糖酵解途径中的一种酶，非常有效：它运行 在扩散控制的极限下。木糖异构酶是最多的 广泛使用的工业酵素，对食品至关重要 工业，而且效率非常低。它的运行速度慢了100,000倍 而不是蒂姆。然而，这两种酶催化了相同的化学转化， 醛和酮的相互转化。了解 TIM的催化效率很重要，因为有一个可怕的人类 蒂姆缺乏症。这是一种先天的新陈代谢错误，遗传于 一种自体隐性遗传方式，导致急性溶血性贫血， 严重的神经功能障碍，感染的易感性增加， 以及心源性猝死的倾向。几位患者的皮损 似乎是一种名为Glu 104的单一氨基酸对天冬氨酸的突变 酸。这项提议的具体目的之一是复制这个人类 TIM在酵母TIM中的突变体，研究了TIM的动力学和稳定性 改变蛋白质，并确定其三维结构。比较 这种结构与野生型酶的结构可能导致低于- 这种替换如何导致致命的遗传性新陈代谢 疾病。其他具体目标侧重于了解特定的 氨基酸对两种酶的催化活性。其中的残留物 问题将被定点突变和属性改变 突变的蛋白质将被确定。TIM系统的一个优点 就是可以确定反应的完整自由能分布 由任何有趣的突变体催化，所以精确的微步骤受 这种突变可以被发现。将获得晶体结构 复合体中的每个突变体都有一种紧密结合的抑制剂，这是一种类似的 反应中的中间体。对于XYI来说，每个变种人都将是 其特点是它对两个独立的阶段的影响 反应：催化糖环开环和异构化。每个 还将对突变株进行结晶学检查，与 实际底物葡萄糖。