CHORISMATE MUTASE--MECHANISM OF A NOVEL ENZYMIC REACTION

本研究的目的是阐明其化学机制。 双功能酶分支酸变位酶预苯酸酯的研究 脱氢酶(CMPDH)。因为CMPDH是一种特殊的酶， 酪氨酸的从头合成，一种不存在的途径 在人类中，对其机制的理解可以导致 开发新的抗生素、抑菌剂或除草剂。 长期目标是利用CMPDH的知识 反应机制，以帮助设计新的抗生素，可以 局部用于口腔，以限制牙周炎的生长。 引起牙菌斑、龋齿和牙周的口腔病原体 疾病是老龄化人口中一个日益严重的问题。 ~(13)C核磁共振将首次应用于研究 CMPDH的作用机制。~(13)C核磁共振将允许观察配体 (底物、产物、反应中间体)结合在活性 酶的位置。将制备13C标记的衬底， 主要是通过已建立的酶学方法及其核磁共振 无论是在溶液中还是在与酶结合时，光谱都将是 获得。酶对化学位移和化学位移的影响 ~(13)C标记配体的偶联常数将揭示 底物、产物和反应中间体的结构 被限制在活动地点。对边界的全面描述 将通过使用五种不同的13C标记来获得配体 底物，每个底物标记在两到四个不同的位置 13C。一些13C原子也将被标记上2H，以确保窄 酶结合型物种的标线。 为了优化从~(13)C核磁共振获得的信息 几种互补的酶结合物种的特性 我们会进行研究。物种间的平衡 将使用放射性底物确定活动地点的位置 和酸淬火技术。平衡约束研究将 测定活性中心的化学计量比(S)。分子 将使用生物技术来增加表达 从克隆的TyrA基因中获得CMPDH。CMPDH的提纯将 进行优化。将进行研究，以优化 该酶在适宜13C条件下的稳定性 核磁共振研究。

The goal of this research is to elucidate the chemical mechanism of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase (CMPDH). Because CMPDH is an enzyme peculiar to the de novo biosynthesis of tyrosine, a pathway which is not present in humans, an understanding of its mechanism can lead to the development of new antibiotics, bacteriostatics, or herbicides. The long term objective is to use the knowledge of the CMPDH reaction mechanism to aid in design of new antibiotics which can be used topically in the oral cavity to limit the growth of the oral pathogens responsible for plaque, caries, and periodontal disease which is a growing problem in an aging population. 13C NMR will be applied for the first time to investigate the mechanism of CMPDH. 13C NMR will allow observation of ligands (substrates, products, reaction intermediates) bound at the active site of the enzyme. 13C labelled substrates will be prepared, predominantly by established enzymological methods, and their NMR spectra both free in solution and when bound to the enzyme will be obtained. The effect of the enzyme on the chemical shifts and coupling constants of the 13C labelled ligands will reveal the structures of the substrates, products, and reaction intermediates bound at the active site. A thorough characterization of the bound ligands will be obtained through use of five different 13C labelled substrates, each labelled in two to four different positions with 13C. Some 13C atoms will also be labelled with 2H to ensure narrow lines for enzyme-bound species. To optimize the information obtained from the 13C NMR characterization of enzyme-bound species, several complementary studies will be carried out. The equilibrium between species bound at the active site will be determined using radioactive substrates and acid quench techniques. Equilibrium binding studies will determine the stoichiometry of active site(s). Molecular biological techniques will be used to increase the expression of CMPDH from the cloned tyrA gene. The purification of CMPDH will be optimized. Studies will be carried out to optimize the stability of the enzyme under conditions appropriate for the 13C NMR studies.