CHARACTERIZATION OF UDP-GALACTOPYRANOSE MUTASE

该提议试图提供一种用于表征酶UDP-吡喃半乳糖苷酶的方法，该酶催化从UDP-吡喃半乳糖到UDP-呋喃半乳糖的不利转化。 UDP-呋喃半乳糖随后展示在几种微生物的细胞表面，包括结核分枝杆菌。 然而，它不显示在真核生物的表面上，使该酶成为开发抗菌剂的靶点。 已从三种不同的细菌菌株中克隆出了该酶，并显示其含有黄素腺嘌呤二核苷酸（FAD），并且可能需要NAD（P）H才能发挥活性。 该提案概述了一种策略，使用紫外可见光谱法来确认这些辅因子，并确定其各自的作用，在酶的机制。 还将检查UDP-吡喃半乳糖苷的底物特异性。通过使用UDP-吡喃半乳糖和UDP-呋喃半乳糖的底物类似物，可以探索结合酶的重要功能位置的鉴定。 从这些信息中，洞察到的结合特异性和反应机制的酶操纵和用于合理设计的抑制剂的酶。

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.