SITE DIRECTED MUTAGENESIS OF ASPARTATE AMINO TRANSFERASE

天冬氨酸氨基转移酶的定点诱变

• 批准号: 3288048
• 负责人: JACK F KIRSCH
• 金额: $ 14.54万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1988-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288048
• 关键词: Bacillus cereus; Escherichia coli; aspartate transaminase; beta lactamase; chemical binding; enzyme structure; enzyme substrate; genetic manipulation; nucleic acid sequence

The objective of this project is to study the mechanism of action of the enzyme aspartate aminotransferase by site-directed mutagenesis. About 6 different amino acids at the active site will be replaced in separate experiments. The mutated enzymes will be examined by an appropriate combination of spectroscopic and kinetic methods. The results will provide information on the relative barrier heights facing the intermediates along the reaction pathway, will help to assign the ionizable groups which are responsible for the inflections in the kinetic pH profiles, and will provide information about the roles of several amino acids in stabilizing intermediates or transition states. A significant aspect of this choice of enzyme is the variety of probes that can be used in combination to examine each mutant. The ubiquity of PLP-dependent coenzymes in amino acid metabolism makes it likely that many of the results of this study will have bearing on the broader problem of the mechanism of action of the entire class. An additional goal of this research that is unrelated to the above, is to design a so-called suicide inhibitor for Beta-lactamase, based on the interaction of a postulated active site enzyme carboxylate moiety with the side-chain of Alpha-cyano benzylpenicillin. The ketenimine formed by general base catalyzed abstraction of the Alpha-proton is a very active electrophilic trapping agent. Success with this inhibitor might lead to a new class of pharmacologically useful inhibitors of Beta-lactamases that could be used in conjunction with penicillins or cephalosporins to increase the efficacy of penicillin-based chemotherapy.

该项目的目的是研究该药物的作用机制 通过定点诱变酶天冬氨酸氨基转移酶。 约6 活性位点的不同氨基酸将被单独替换 实验。 突变的酶将通过适当的方法进行检查 光谱法和动力学方法的结合。 结果将提供 有关中间体沿线面对的相对势垒高度的信息 反应途径，将有助于分配可电离基团 负责动态 pH 曲线的变化，并将 提供有关几种氨基酸在稳定中的作用的信息 中间体或过渡态。 此次选择的一个重要方面是 酶是可以组合使用来检查的各种探针 每个突变体。 氨基酸中 PLP 依赖性辅酶的普遍存在 新陈代谢使得这项研究的许多结果可能会 关系到整个行动机制的更广泛问题 班级。 这项研究的另一个与上述无关的目标是 设计一种所谓的β-内酰胺酶自杀抑制剂 假设的活性位点酶羧酸盐部分与 α-氰基苄青霉素的侧链。 烯酮亚胺形成于 α-质子的一般碱催化抽象是非常活跃的 亲电捕获剂。 这种抑制剂的成功可能会导致 新型药理学上有用的 β-内酰胺酶抑制剂 可以与青霉素类或头孢菌素类药物联合使用，以增加 以青霉素为基础的化疗的疗效。