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.
