Uridine diphosphate glucose pyrophosphorylase. Crystallization and properties of the enzyme from rabbit liver and species comparisons.

Rabbit liver UDP-glucose pyrophosphorylase has been crystallized and further purified to a specific activity of 200 by using preparative centrifugation with sucrose density gradients. The pyrophosphorylase requires a divalent cation. Magnesium is preferred, although manganese, cobalt, and calcium can serve as less effective alternate cation activators. Maximum activity requires a reducing agent, and the enzyme has a broad pH range from 7.0 to 10.5. The molecular weight of the enzyme is approximately 400,000, with eight identical subunits. While the enzyme shows no tendency to form multimolecular aggregates as do other liver pyrophosphorylases, the eight subunits are arranged in a stacked tetrameric configuration. Amino acid analysis, peptide mapping, gel electrophoresis, and sedimentation showed the enzyme to be homogeneous with a single NH*-terminal amino acid being serine. The subunits of the rabbit liver enzyme are chemically identical as confirmed by the foregoing analyses. Although UDP-glucose is the most active substrate, the enzyme will catalyze the pyrophosphorylation of a variety of nucleoside diphosphate hexoses. Rates as high as 14% of that for UDP-glucose were obtained using UDP-galactose as a substrate. The ratio of glucose to galactose activity remained constant throughout purification and no evidence was found for a separate enzyme for UDP-galactose. The equilibrium constant in the direction of UDP-glucose formation was 0.16. The apparent Michaelis constants for UDP-glucose, UTP, and glucose l-phosphate were 6.6 x 10p5, 3.8 x 10e5, and 4.6 x lo”, respectively, and that for UDP-galacfose was 4.2 x 10m4. UDP-galactose and galactose 1 -phosphate are competitive inhibitors of UDP-glucose and glucose l-phosphate with apparent constants of 2.2 x 1O-4 and 7.3 x 10m3, respectively. Either 8 moles of UDPglucose or UDP-galactose were bound per mole of enzyme, and the bound UDP-galactose could be replaced stoichiometrically by equivalent quantities of UDP-glucose.