UDP-Galactose 4-Epimerase from Kluyveromyces fragilis: Substrate Specific Inactivation during Catalytic Turn Over

Abstract

UDP-Galactose 4-epimerase, an enzyme with bound NAD, reversibly converts UDP-Galactose (UDP-Gal) to UDP-Glucose (UDP-Glc). This enzyme from Kluyveromyces fragilis was inactivated during the conversion of UDP-Gal to UDP-Glc by 30min under standard assay conditions, while it remained active for over 4h during the reverse reaction. The rate of inactivation and reduction of the bound NAD to NADH were similar. The rate of inactivation and formation of enzyme-bound NADH were dependent on the concentrations of enzyme and UDP-Gal. After complete inactivation, no further NADH fluorescence could be generated. Rate of inactivation of epimerase with increasing UDP-Gal concentration followed a linear dependency. Differences in the conformational changes at the catalytic sites imparted by UDP-Gal and UDP-Glc were evident from the patterns of thermal inactivation of the complexes of epimerase with substrate analogs. With saturating concentration of UDP-Gal, the Arrhenius energy of activation (Ea) during inactivation was found to be nearly zero. The favorable interaction of UDP-Gal with epimerase was further confirmed by analyzing the X-ray crystallographic structure and molecular modeling studies of epimerase from a related species, Saccharomyces cerevisiae. Thus, the high rates of formation and dissociation of the epimerase and UDP-Gal complex seems to impart and release of stress on the enzyme leading to its inactivation.