ENZYMES OF THE HISTIDINE BIOSYNTHETIC PATHWAY

长期目标建立一个详细的了解 参与组氨酸生物合成途径的酶促步骤，导致 最终了解酶活性位点的组织， vivo. 本研究的一个重点是描述主要的机制， 途径中三个催化步骤的各个方面，并将其与 蛋白质结构的信息。 医学相关性。 知识 从这些研究中获得的信息最终将对设计至关重要， 选择新的抗菌剂、抗真菌剂或除草剂， 氨基酸和嘌呤代谢中的主要事件。 因为 研究中代谢中存在的独特化学特征， 针对这些步骤的抑制剂将通过一种新的 行动方式。 这一特点在 开发对病原体具有选择性的抗真菌剂， 对宿主无毒。 具体目标1. 的机理研究 由5 '-ProFAR异构酶催化的酶促Amadori重排将是 继续使用氢交换实验，底物和产物 类似物作为抑制剂。 适当的初步调查将是 建立了一种方法，以达到三维 使用NMR和/或X射线衍射技术分析酶的结构。 2. 介导谷氨酰胺的关键亚基相互作用 咪唑甘油磷酸合酶的酰胺转移酶性质 将使用新的体内随机诱变筛选来发现。 3. 一 咪唑甘油磷酸酯催化反应序列的研究 将进一步研究合成酶。 该提案的一个主要目标是 发生在细胞表面的碳-氮连接反应的鉴定 这种酶通过使用快速淬灭动力学和突变酶， 能够部分翻转。 4. 急诊磷酸核糖AMP 由双功能hisIE基因编码的环化水解酶将被纯化， 用于模拟合成。 这种酶的底物的制备 将使用编码突变hisIE的重组DNA系统进行改进 基因 咪唑甘油磷酸酯的三维结构 将使用X射线测定来自新型隐球菌的葡萄糖转移酶 结晶学 酶的紧密结合竞争性抑制剂将 用于共结晶以鉴定活性位点及其 与金属结合位点的关系。

Long term objectives To establish a detailed understanding of the enzymatic steps involved in the histidine biosynthetic pathway, leading ultimately to a knowledge of the organization of enzyme active sites in vivo. A focus of this investigation is to delineate the major mechanistic aspects of three catalytic steps in the pathway, and to relate this information to their protein structures. Medical Relevance. The knowledge gained from these studies will ultimately be critical for the design and selection of new antibacterial, antifungal or herbicidal agents targeted at primary events in a amino acid and purine metabolism. Because of the unique chemical features present in the metabolism under investigation, inhibitory agents directed at these steps would be operating by a novel mode of action. This feature is of particular importance in the development of antifungal that would be selective for a pathogen and nontoxic to a host. Specific Aims 1. The mechanistic studies of the enzymatic amadori rearrangement catalyzed by 5'-ProFAR isomerase will be continued using hydrogen exchange experiments, substrate and product analogs as inhibitors. The appropriate preliminary investigations will be conducted to establish a method for arriving at a three dimensional structure of the enzyme using NMR and/or x-ray diffraction techniques. 2. The critical subunit interactions that mediate the glutamine amidotransferase properties of the imidazoleglycerol phosphate synthase will be discovered using a novel in vivo random mutagenesis screen. 3. A study of the sequence of reactions catalyzed by imidazoleglycerol phosphate synthase will be further examined. A major goal of this proposal will be the identification of the carbon-nitrogen ligation reactions which occur on this enzyme by using rapid-quench kinetics and mutant enzymes that are capable of partial turnover. 4. The E. coli phosphoribosyl-AMP cyclohydrolase encoded by the bifunctional hisIE gene will be purified for use in analog syntheses. A preparation of the substrate for this enzyme will be refined using a recombinant DNA system that encodes a mutant hisIE gene. The three dimensional structure of imidazoleglycerol phosphate dehydratase from Cryptococcus neoformans will be determined using x-ray crystallography. A tight-binding competitive inhibitor of the enzyme will be used in co-crystallizations to identify the active site and its relationship to the metal binding site.