CHARACTERIZATION OF UDP-GALACTOPYRANOSE MUTASE

本建议试图提供一种表征UDP-半乳糖变位酶的方法，该酶催化UDP-半乳糖向UDP-呋喃半乳糖的不利转化。随后，UDP-半乳呋喃糖被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，使该酶成为抗菌剂开发的目标。该变位酶是从三个不同的细菌菌株中克隆出来的，它含有一个黄素腺嘌呤二核苷酸(FAD)，可能需要NAD(P)H才能发挥活性。这项建议概述了一种使用紫外可见光谱来确认这些辅因子并确定它们在酶机制中的各自作用的策略。还将检测UDP-半乳糖变位酶的底物特异性。通过使用UDP-半乳糖和UDP-呋喃半乳糖的底物类似物，可以探索与酶结合的重要功能位置。通过这些信息，洞察变位酶的结合特异性和反应机理，可以操纵和使用该酶的抑制剂的合理设计。

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.