PURINE CYCLOHYDROLASE OF HISTIDINE BIOSYNTHESIS

植物和微生物中组氨酸的生物合成是通过一种 利用高级代谢前体的独特代谢途径 例如ATP和5-磷酸-D-核糖基-1-焦磷酸(PRPP)作为碳 和氮源。由于这一途径在哺乳动物中不存在， 所提出的研究在Rational中具有重要应用 抗菌、抗真菌、抗真菌酶抑制剂的研究进展 除草剂应用。丰富的序列比对数据表明 该酶的相似性和进化关系 古细菌、真细菌和酵母。因此，这一行动的具体目的是 建议对N1-磷酸核糖基-AMP环水解酶进行表征 (HISI)，一种酶，催化关键和独特的一步 组氨酸的生物合成：三磷酸腺苷的嘌呤环的水解。 我们将从分析具体要求开始(例如，金属 离子)，以及单官能团HISI的稳态动力学性质 使用一种特殊的连续UV-Vis检测方法。这个 氨基酸序列比对数据和化学分析结果 将使用修饰实验来选择特定的氨基酸 作为聚合酶链式反应介导的位点特异性突变的候选基因 以确定与催化有关的特定残基。脱氨基 提出了N1-磷酸核糖基-AMP的类似物作为潜在的用途 抑制剂和机械探头。更复杂的研究， 双功能大肠杆菌HisIE酶也将在一旦 了解单个功能HISI的细节。潜力 这些比较研究对酶的进化的意义 催化是耐人寻味的。电喷雾质谱仪，18-O 将使用标记研究和UV-Vis来调查 这种酶的作用机制。这项研究的长期目标是 将HISI作为特定设计的潜在目标进行评估 组氨酸生物合成的抑制剂。

Histidine biosynthesis in plants and microorganisms occurs via an exclusive metabolic pathway that employs advanced metabolic precursors such as ATP and 5-phospho-D-ribosyl-1-pyrophosphate (PRPP) as carbon and nitrogen sources. Since this pathway is not present in mammals, the proposed research has important applications in the rational development of enzyme inhibitors with antibacterial, antifungal, and herbicidal applications. Abundant sequence alignment data indicate the similarity and evolutionary relationship of this enzyme in archaebacteria, eubacteria, and yeast. Thus, the specific aim of this proposal is to characterize N1-phosphoribosyl-AMP cyclohydrolase (HisI), an enzyme that catalyzes a critical and unique step in the biosynthesis of histidine: the hydrolysis of the purine ring of ATP. We will start by analyzing the specific requirements (e.g. metal ions), and steady state kinetic properties of monofunctional HisI from Methanococcus vannielii using a specific continuous UV-vis assay. The amino acid sequence alignment data and results of chemical modification experiments will be used to select specific amino acids as candidates for PCR mediated site specific mutagenesis in an effort to identify specific residues involved in catalysis. A desamino analog of N1-phosphoribosyl-AMP is proposed for use as potential inhibitor and mechanistic probe. Studies with the more complex, bifunctional E. coli HisIE enzyme will also be pursued once the details of the single function HisI are understood. The potential implication of these comparative studies into the evolution of enzyme catalysis is intriguing. Electrospray mass spectrometry, 18-O labeling studies and UV-vis will be employed in the investigation of the mechanism of this enzyme. A long range goal of this study is to evaluate HisI as a potential target for the design of specific inhibitors of histidine biosynthesis.