Co-purification and characterization of UDP-glucose 4-epimerase and UDP-N-acetylglucosamine 4-epimerase from porcine submaxillary glands.

UDP-glucose 4-epimerase and UDP-N-acetylglucosamine 4-epimerase have been co-purified about 9,000-fold from porcine submaxillary glands by affinity chromatography on UDP-hexanolamine-agarose. The homogeneous epimerase has apparent Mr = 88,000 and contains two subunit species with apparent Mr = 37,000 and 35,000, respectively. The two subunits, however, are indistinguishable as judged by peptide mapping. The purified enzyme catalyzes equally well the reversible reactions UDP-glucose in equilibrium UDP-galactose and UDP-N-acetylglucosamine in equilibrium UDP-N-acetylgalactosamine. At saturating substrate concentrations, the ratio of the rate of the former reaction to that of the latter is 1.13 in the forward direction and 0.44 in the backward direction. Both reactions have the same Keq = 0.38 and the same dependence on pH. Moreover, both activities are lost at about the same rate by heat denaturation of the epimerase or reaction with N-ethylmaleimide. Kinetic analysis reveals that the reactants for one reaction are competitive inhibitors of the other reaction, with the Ki values of the inhibitors essentially identical with their Km values as substrates. Taken together, these studies suggest that UDP-glucose 4-epimerase and UDP-N-acetylglucosamine 4-epimerase activities reside in a single enzyme.