ENZYMATIC ANALYSIS OF SUCCINYL TRANSFER

琥珀酰转移的酶分析

• 批准号: 2777591
• 负责人: TIMOTHY L BORN
• 金额: $ 3.67万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-11 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2777591
• 关键词: X ray crystallography; acidity /alkalinity; active sites; acyltransferase; chemical kinetics; crystallization; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; expression cloning; molecular cloning

DESCRIPTION Homoserine transsuccinylase (HTS) catalyzes the conversion of homoserine to U- succinylhomoserine. This is the first specific step in the biosynthesis of methionine and S-adenosylmethionine by microorganisms. Due to the critical and specific functions in bacterial amino acid and co- factor biosynthesis, enzymes in this pathway present potential targets for broad spectrum antibacterial inhibitor design. Since the initial submission of this proposal, E. coli HTS has been cloned, over-expressed, and purified. The kinetic mechanism of HTS will be determined by a series of experiments. Experiments using variable concentrations of both substrates indicate that HTS employs a ping-pong mechanism. DTNP inhibits the enzyme, suggesting the presence of an active site thiol. We will investigate this possibility by labeling HTS with thiol modifying agents, such as vinyl pyridine, and identifying a labeled peptide. A detailed pH profile will be conducted to determine the ionization behavior of the group(s) that are involved in catalysis or substrate binding that are involved in catalysis or substrate binding. Initial protein crystallization attempts have identified conditions which produce microcrystals. We will optimize these conditions in an effort to grow diffraction quality crystals for three-dimensional structure determination. The crystals will be soaked with various substrates and inhibitors, which will define the active site and delineate important interactions. Time permitting, we will attempt to clone the functionally related protein, homoserine transacetylase, from either H. influenza or M. tuberculosis in order to conduct a comparative study between the two acyl transferases.

描述 高丝氨酸转琥珀酰酶（HTS）催化高丝氨酸 变成U-琥珀酰高丝氨酸。这是第一个具体步骤， 通过微生物生物合成甲硫氨酸和S-腺苷甲硫氨酸。由于 细菌氨基酸和辅酶的关键和特殊功能， 因子生物合成，该途径中的酶提供了 广谱抗菌抑制剂设计。由于初始 该提案的提交，E。coliHTS已被克隆，过量表达， 并被净化高温超导的动力学机制将由一系列的 的实验。实验使用可变浓度的 衬底表明，HTS采用乒乓机制。DTNP抑制 该酶，表明存在活性位点硫醇。我们将 通过用硫醇修饰剂标记HTS来研究这种可能性， 如乙烯基吡啶，并鉴定标记的肽。详细的pH值 将进行曲线以确定 参与催化或底物结合的基团， 参与催化或底物结合。初始蛋白 结晶尝试已经确定了产生 微晶我们将优化这些条件， 用于三维结构的衍射质量晶体 保持战略定力晶体将被浸泡在各种基质中， 抑制剂，这将定义活性位点，并描绘重要的 交互.如果时间允许，我们将尝试克隆功能 高丝氨酸转乙酰酶（homoserine transacetylase）。流感或M. 结核病，以进行比较研究之间的两个酰基 转移酶