SITE DIRECTED MUTAGENESIS OF ASPARTATE AMINO TRANSFERASE

本项目的目的是研究 酶天冬氨酸氨基转移酶的定点突变。 约6 活性位点上的不同氨基酸将被单独替换， 实验 突变的酶将通过适当的 光谱和动力学方法的组合。 结果将提供 关于中间体沿着面对的相对势垒高度的信息 反应途径，将有助于分配电离基团， 负责动力学pH曲线的拐点，并将 提供关于几种氨基酸在稳定 中间体或过渡态。 这一选择的一个重要方面是 酶是各种探针，可以组合使用，以检查 每个变种人 PLP依赖性辅酶在氨基酸中的普遍性 代谢使得这项研究的许多结果可能会有 影响到更广泛的问题的整个行动机制 课 这项研究的另一个目标与上述无关，是 设计一种所谓的β-内酰胺酶自杀抑制剂， 假定的活性位点酶羧酸酯部分与 α-氰基苄青霉素侧链。 由以下形成的烯酮亚胺： 一般碱催化的α-质子的提取是一种非常活跃的 亲电捕集剂 这种抑制剂的成功可能会导致 一类新的非常有用的β-内酰胺酶抑制剂， 可与青霉素或头孢菌素联合使用， 以青霉素为基础的化疗的疗效。

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.