This proposal outlines a strategy for investigating the mechanism of an unusual enzymatic transformation: the conversion of UDP-galactopyranose (UDP-galp) to UDP-galactofuranose (UDP-galf) via a novel ring contraction catalyzed by the bacterial enzyme UDP-galactopyranose mutase. Since the enzyme converts UDP-galp to UDP-galf in low amounts (5-8 percent) at equilibrium, we propose to study the conversion in the reverse direction, from UDP-galf to UDP-galp. This will be accomplished by chemically synthesizing UDP-galf. This will permit us to more accurately monitor the conversion because we will assay for formation of UDP-galp, the thermodynamically favored product. The chemical synthesis of UDP-galf will also allow us to confirm the structure of the reported product of the enzymatic reaction. We also plan to investigate different mechanistic proposals by synthesizing and testing substrate analogs of UDP-galf, designed to probe different aspects of these mechanisms. Finally, we plan to investigate the development of a continuous linked-enzyme spectrophotometric assay as an alternative to the currently used HPLC assay.
