Purification and properties of [formula omitted] 2′-epimerase from rat liver

Abstract

1. 1. UDP-N- acetylglucosamine 2′-epimerase (EC 5.1.3.7) has been purified 500-fold from rat liver with UDP as stabilizing agent. In the presence of UDP and dithiothreitol, the final preparation is stable and loses only 20% of the initial activity over 3 days storage at 4 °C. 2. 2. UDP and UDP-N- acetylglucosamine protect 2′-epimerase from inactivation by aging. UDP also is a competitive inhibitor, the K i being 0.14 mM. Uridine, the stabilizing agent used by previous workers, neither stabilized nor inhibited the enzyme. It is suggested that UDP interacts with the enzyme at the substrate site. Dithiothreitol also stabilizes the enzyme but only under limited conditions. 3. 3. 2′-Epimerase exhibits negative cooperativity in UDP-N- acetylglucosamine binding. The apparent K m value in the lower activity range is 0.08 mM. The negative cooperativity is abolished by UDP or CMP-N- acetylneuraminic acid. 4. 4. 2′-Epimerase is highly sensitive to inhibition by CMP-N- acetylneuraminic acid; the concentration required for 50% inhibition (0.025 mM) is close to its intracellular levels. A Hill plot yielded the Hill coefficient ( n H ) of 5.7. 5. 5. When UDP but not dithiothreitol is present, the purified enzyme undergoes alterations in UDP-N- acetylglucosamine binding such as the disappearance of negative cooperativity, an increase in K m and the appearance of substrate inhibition; all are reversed by dithiothreitol.