UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of
UDP-
galactopyranose to
UDP-galactofuranose, the precursor of galactofuranose (Galf). Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of
Chagas' disease. Galf) is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the
flavin must be in the reduced state for activity and the exact role of the
flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that
NADPH is an effective redox partner of TcUGM. The substrate,
UDP-
galactopyranose, protects the
enzyme from reacting with molecular
oxygen allowing TcUGM to turnover ∼1000 times for every
NADPH oxidized. Spectral changes consistent with a
flavin iminium ion, without the formation of a
flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the
flavin acting as a nucleophile. In support of this role, a
flavin-
galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented.