CHARACTERIZATION OF UDP-GALACTOPYRANOSE MUTASE

本提案试图提供一种表征酶的方法，UDP-半乳糖酰脲酶，它催化从UDP-半乳糖酰脲糖到UDP-半乳糖呋喃糖的不利转化。udp -半乳糖呋喃糖随后显示在包括结核分枝杆菌在内的几种微生物的细胞表面。然而，它不显示在真核生物的表面，使酶的抗菌药物的发展目标。从三种不同的菌株中克隆出了这种突变酶，发现它含有一种黄素腺嘌呤二核苷酸（FAD），可能需要NAD(P)H才能产生活性。本文提出了一种利用紫外可见光谱学来确认这些辅助因子并确定它们在酶促机制中的各自作用的策略。udp -半乳糖酰脲酶的底物特异性也将被检查。通过使用UDP-半乳糖酰脲糖和UDP-半乳糖呋喃糖的底物类似物，可以探索与酶结合的重要功能位置。从这些信息中，可以深入了解突变酶的结合特异性和反应机制，并用于合理设计该酶的抑制剂。

This proposal attempts to provide a method for characterizing the enzyme, UDP-galactopyranose mutase, which catalyzes the unfavorable conversion from UDP-galactopyranose to UDP- galactofuranose. UDP-galactofuranose is subsequently displayed on the cell surface of several microbes including Mycobacterim tuberculosis. It is not, however, displayed on the surface of eukaryotic organisms, making the enzyme a target for the development of antimicrobial agents. The mutase has been cloned from three different bacterial strains and shown to contain a flavin adenine dinucleotide (FAD) and possibly require NAD(P)H for activity. This proposal outlines a strategy using UV-visible spectroscopy to confirm these cofactors and determine their respective roles in the enzymatic mechanism. Substrate specificity of UDP-galactopyranose mutase will also be examined. By using substrate analogs of both UDP-galactopyranose and UDP- galactofuranose, identification of the important functional positions in binding to the enzyme can be explored. From this information, insight into the binding specificity and reaction mechanism of the mutase can manipulated and used for the rational design of inhibitors for the enzyme.