SITE-SPECIFIC MUTAGENESIS OF ISOMERASES

这个项目的目标是使用一个组合的网站导向 诱变和X射线晶体学来了解结构基础 酵母磷酸丙糖异构酶（TIM）的催化能力， 阐明木糖异构酶（也称为葡萄糖）的作用机制 异构酶，缩写为XyI）。 磷酸丙糖异构酶是 糖酵解途径中的酶，是非常有效的：它的运作 在扩散控制的极限。 木糖异构酶是最 广泛使用的工业酶，对食品至关重要 工业，而且效率很低。 它的运行速度要慢十万倍 比TIM 但这两种酶催化的是相同的化学转化， 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 TIM缺乏症。 这是一种先天性的代谢缺陷， 是一种常染色体隐性遗传，导致急性溶血性贫血， 严重的神经功能障碍，增加感染的易感性， 以及心源性猝死的倾向。 几个病人的病变 似乎是单个氨基酸Glu 104突变为天冬氨酸 酸 这项提案的具体目标之一是复制这个人类 酵母TIM中的TIM突变体，表征该突变体的动力学和稳定性。 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的差异可能导致 这种替代是如何导致致命的遗传代谢的 疾病 其他具体目标侧重于了解特定的 氨基酸在两种酶的催化活性。 中的残基 这个问题将通过定点诱变和 将确定突变蛋白质。 TIM系统的一个优点是 可以确定反应的完整自由能分布 催化任何有趣的突变，所以确切的微步影响， 突变是可以被发现的。 将获得晶体结构， 每一个突变体都与一种类似的紧密结合抑制剂复合， 反应中的中间体。 对XyI来说，每一个突变体 其特点是对两个不同阶段的影响， 反应：糖环的催化开环和异构化。 每 突变体也将进行晶体学检查，与 实际底物葡萄糖。

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.