ENZYMATIC ANALYSIS OF SUCCINYL TRANSFER

描述 高丝氨酸转移琥珀酸酶(HTS)催化高丝氨酸转化 到U-琥珀酰高丝氨酸。这是 微生物生物合成蛋氨酸和S-腺苷蛋氨酸到期 对细菌氨基酸和辅酶的关键和特定功能的研究 因子生物合成，这一途径中的酶是潜在的靶标 广谱抗菌抑制剂设计。从最初的 提交本提案后，大肠杆菌HTS已被克隆、过度表达、 并得到净化。高温超导的动力学机制将通过一系列研究来确定 实验的结果。使用不同浓度的两者进行实验 衬底表明，HTS采用乒乓球机制。DTNP抑制 该酶，表明存在活性部位硫醇。我们会 通过用硫醇修饰剂标记HTS来研究这种可能性， 例如乙烯基吡啶，并鉴定标记的多肽。详细的PH值 将进行剖面图以确定电离行为 参与催化或底物结合的组(S) 参与催化或底物结合的。初始蛋白质 结晶尝试已经确定了产生 微晶。我们将优化这些条件，努力实现增长 具有衍射性的三维结构晶体 决心。晶体将被各种底物浸泡， 抑制剂，它将定义活性部位并描绘出重要的 互动。如果时间允许，我们将尝试从功能上克隆 相关的蛋白质，高丝氨酸转乙酰基酶，来自流感病毒或流感病毒。 结核分枝杆菌两个酰基之间的比较研究 转移酶。

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.