SITE SPECIFIC MUTAGENESIS OF ISOMERASES

本提案的目的是使用现场指导技术， 诱变，结合X射线晶体学，以了解酶如何 能够有效地催化异构化反应。 异构化 是最简单的代谢反应，催化它们的酶是 许多遗传性代谢疾病的重要病原体。 在本提案中，选择了三种异构酶作为研究系统： 糖酵解酶磷酸丙糖异构酶 磷酸葡萄糖异构酶（与淋巴因子相同） 神经白细胞介素）和重要的食品加工酶木糖异构酶 (also称为葡萄糖异构酶）。 所有这些酶都催化一种 底物/单一产物平衡，其允许直接 任何酶-底物复合物的晶体学观察 变种人 对于磷酸丙糖异构酶，该提案的具体目标是 了解保守氨基酸在活性氨基酸中的作用， 位置，并剖析底物诱导构象的机制， 导致灵活的环路屏蔽活动站点免受大容量 溶剂后 了解磷酸丙糖的作用机制 异构酶有望增加对严重疾病的理解， 当突变导致这种酶的次优量时会产生结果。 对于木糖异构酶，具体的目的是了解酶如何 催化糖环打开，以了解如何在两个金属离子， 活性位点在氢化物转移机制中协同作用， 该酶利用磷酸丙糖异构酶的质子转移机制。 这种改变的木糖异构酶将具有很大的价值， 生物技术. 对于磷酸葡萄糖异构酶，具体目标是表达猪 肌肉酶在E.大肠杆菌，确定其晶体结构， 底物结合，并确定其机制的重要驻留， 其包括开环，随后是碱催化的质子转移。 由于磷酸葡萄糖异构酶与淋巴因子相同 神经白细胞介素，其结构和作用机制的知识将有 对神经系统疾病的理解产生了重大影响， 艾滋病相关的痴呆症，其中这种酶已被证明参与。

The objective of this proposal is to use the technique of site-directed mutagenesis, combined with X-rays crystallography, to learn how enzymes are able to catalyze isomerization reactions efficiently. Isomerizations are the simplest metabolic reactions, and enzymes that catalyze them are important causative agents of a number of inherited metabolic diseases. Three isomerases have been chosen as systems to study in this proposal: the glycolytic enzyme triosephosphate isomerase, the glycolytic enzyme phosphoglucose isomerase (which is identical to the lymphokine neuroleukin), and the important food-processing enzyme xylose isomerase (also known as glucose isomerase). All of these enzymes catalyze a single substrate/single product equilibration, which allows direct crystallographic observation of the enzyme-substrate complex for any mutant. For triosephosphate isomerase, the specific aims of the proposal are to understand the roles of conserved amino acids in and away from the active site, and to dissect the mechanism of the substrate-induced conformational changes that causes a flexible loop to shield the active site from bulk solvent. Knowledge of the mechanism of action of triosephosphate isomerase is expected to add to understanding of the severe disease that results when mutations lead to a suboptimal amount of this enzyme. For xylose isomerase, the specific aims are to learn how the enzyme catalyzes sugar ring opening, to understand how the two metal ions in the active site cooperate in the hydride transfer mechanism, and to reengineer the enzyme to use the triosephosphate isomerase proton transfer mechanism. Such an altered xylose isomerase would be of great value for biotechnology. For phosphoglucose isomerase, the specific aims are to express the pig muscle enzyme in E. coli, determine its crystal structure with and without substrate bound, and identify the resides important for its mechanism, which involves ring opening followed by base-catalyzed proton transfer. Since phosphoglucose isomerase is identical to the potent lymphokine neuroleukin, knowledge of its structure and mechanism of action will have a significant impact on understanding of neurological disorders such as AIDS-related dementia, in which this enzyme has been shown to be involved.