Uridine Diphosphate Glucose Pyrophosphorylase

Abstract

Abstract A purification and crystallization procedure has been developed for uridine diphosphate glucose pyrophosphorylase from human liver. The 500-fold purified enzyme was found to be almost homogeneous by polyacrylamide gel electrophoresis and sedimentation velocity. The crystalline enzyme has an absolute requirement for divalent cations. Magnesium at 3.0 mm and manganese at 1.0 mm gave about equal and maximum activation while cobalt at 1.0 mm resulted in 40% activation of the pyrophosphorylase. The pH optimum is broad, ranging between 7.6 and 9.2. In the direction of uridine diphosphate glucose synthesis, the equilibrium constant is 0.15. The apparent Michaelis constants from Lineweaver-Burk plots for uridine diphosphate glucose, glucose 1-phosphate, and uridine triphosphate are 5.0 x 10-5, 9.5 x 10-5, and 4.8 x 10-5 m, respectively. With the pyrophosphorylase, sigmoidal kinetics was observed when the reaction was initiated with inorganic pyrophosphate. The slope of the Hill plot decreased from 2.5 to 1.5 when the enzyme was previously incubated in 2 mm inorganic pyrophosphate for 5 min. The apparent Km for inorganic pyrophosphate was estimated to be 2.1 to 2.6 x 10-4 m. Uridine diphosphate is a competitive inhibitor of uridine diphosphate glucose. The enzyme was not specific for either nucleoside or the hexose component of the nucleoside diphosphate hexose. At nonlimiting substrate concentrations, the activity ratio of the pyrophosphorylase with uridine diphosphate glucose and uridine diphosphate galactose as substrates remained constant throughout the purification and crystallization procedures. Thus, human liver does not appear to contain a separate uridine diphosphate galactose pyrophosphorylase.