SITE DIRECTED MUTAGENESIS OF ASPARTATE AMINO TRANSFERASE

本项目的目的是研究黄连素的作用机制。 酶天冬氨酸氨基转移酶的定点突变。大约6个 活性部位的不同氨基酸将被分别替换 实验。突变的酶将由适当的 光谱方法和动力学方法的结合。结果将提供 中间体沿线面临的相对障碍高度的信息 反应途径有助于分配可电离基团，这些基团是 对动态pH曲线的变化负责，并将 提供关于几种氨基酸在稳定中的作用的信息 中间体或过渡态。这一选择的一个重要方面 酶是可以组合使用的各种探针，用于检测 每个变种人。依赖PLP的辅酶在氨基酸中的普遍存在 新陈代谢使得这项研究的许多结果很可能会 关系到更广泛的问题，即整个 班级。 这项研究的另一个与上述无关的目标是 在此基础上，设计一种所谓的β-内酰胺酶自杀抑制剂 一个假定的活性部位酶羧酸部分与 α-氰基青霉素的侧链。酮亚胺由以下物质形成 总碱催化的α-质子抽提是一种非常活跃的 亲电性捕捉剂。这种抑制剂的成功可能会导致 一类新的药理有用的β-内酰胺酶抑制剂 可与青霉素或头孢菌素联合使用以增加 青霉素为主的化疗的疗效观察。

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.