Rate Enhancements Brought About by Uridine Nucleotides in the Reduction of NAD+ at the Active Site of UDP-Galactose 4-Epimerase

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP glucose. In the course of the reaction, the galacto- and glucopyranosyl rings undergo reversible oxidation to the 4-keto-glucopyranosyl ring by reaction with the enzyme-bound NAD+. The UDP-moiety of a substrate participates in catalysis by inducing a conformational change in the enzyme that enhances the chemical reactivity of NAD+ toward reducing agents. This is modeled by UMP-dependent reductive inactivation of the epimerase-NAD+ complex by various sugars as well as by borohydrides. The present work shows that UDP also activates the reduction of epimerase-bound NAD+. Furthermore, the reduction of epimerase-NAD+ by glucose at a very slow rate can be observed under anaerobic conditions in the absence of a uridine nucleotide. Comparisons of the second order rate constants for reduction of epimerase-NAD+ by glucose in the presence and absence of uridine nucleotides have allowed the magnitude of the rate enhancements brought about by UMP and UDP to be estimated. The rate enhancements by UMP and UDP correspond to decreases of 5.7 and 4.1 kcal mol−1, respectively, in the activation energy. A decrease of 4.0 kcal mol−1 in the activation energy for reduction by NaBH3CN was brought about by UMP-binding. The maximum increases in the reduction potential of epimerase-NAD+ induced by UMP- and UDP-binding are estimated to be 120 and 90 mV, respectively. The results are well correlated with the perturbations of the nicotinamide-13C NMR chemical shifts brought about by uridine nucleotides (Burke, J. R., and Frey, P. A. (1993) Biochemistry32, 13220–12230).