4-Uloses as Intermediates in Enzyme-Nicotinamide Adenine Dinucleotide-mediated Oxidoreductase Mechanisms

UDP-galactose-4-epimerase and TDP-glucose-oxidoreductase are shown to have analogous reaction mechanisms. TDP-glucose is a substrate for both enzymes, and when labeled with tritium at carbon 4 it displayed an isotope effect for both enzymes. Neither enzyme exhibited an isotope effect with the 3-tritiated substrate. The value of the isotope effect for the 4-tritiated substrate was identical for both enzymes within the limits of error. Consistent with these findings, TDP-[4-3H]glucose donated tritium to enzyme-bound NAD+, leading to formation of enzyme-[3H1]NADH. This suggests that a nucleoside diphospho-4-ulose (of the configuration d-xylo-4-hexosulose) is an intermediate in the reactions catalyzed by UDP-galactose-4-epimerase and TDP-glucose-oxidoreductase.