MECHANISM OF ACTION OF UDPGal-4-EPIMERASE: ISOTOPE EFFECT STUDIES

The synthesis of uridine diphosphate glucose and uridine diphosphate galactose labeled with tritium in the 4-position permits the observation of the isotope effect associated with the UDPGal-4-epimerase reaction. This isotope effect has been measured for the reaction proceeding in both directions and the values of k T /k H fall in the range 1.5 to 3.0. This allows certain conclusions to be drawn concerning the mechanism of this important enzymatic catalysis. The direction and magnitude of the effect indicate that the 4-hydrogen is removed from the hexose in the course of the reaction. They also appear to dispose of several mechanisms that have been proposed for the epimerase. Specifically, mechanisms involving cleavage of the CO or CC bonds at carbon-4 of the hexose moiety, such as cleavage of the carbon chain, elimination and readdition of the carbon-4 hydroxyl as water, or ionization to form a carbonium ion, are not supported by the observed data. A mechanism consistent with all observations involves transfer of the hydrogen at carbon-4 to the enzyme in a step that is not rate determining. This is followed immediately by the rate-determining step, which may well be the reorganization of the enzyme-substrate complex to allow return of the hydrogen in the opposite configuration. The larger estimate of the isotope effect indicates a transfer of the carbon-4 hydrogen to a nitrogen atom located in the enzyme's structure; the smaller estimate is consistent with transfer to oxygen or carbon. Transfer to sulfur appears to be definitely eliminated.During this work a degradative procedure that will permit the location and quantitation of the carbon-bound tritium in any hexose or pentose was developed.