Crystallization and Properties of Uridine Diphosphate Glucose Pyrophosphorylase from Liver

Abstract

Abstract The purification and crystallization of uridine diphosphate glucose pyrophosphorylase from calf liver has been described. As much as 0.3% of the extractable protein is the pyrophosphorylase, which catalyzes the synthesis of uridine diphosphate glucose. After recrystallizing the enzyme twice, constant specific activity is attained; it appears to be almost homogeneous on polyacrylamide gel electrophoresis and on ultrasedimentation. The recrystallized enzyme is not highly specific for either the nucleoside or the sugar component of the nucleoside diphosphate sugar. When thymidine, cytidine, or guanosine are substituted for uridine, or when galactose, mannose, or xylose are substituted for glucose, the resulting compounds have from 0.1 to 4% of the activity of uridine diphosphate glucose. The molecular weight of the recrystallized enzyme is about 400,000; the specific activity, 240; and the turnover number, 83,000. The following Km values were obtained for each substrate: glucose 1-phosphate, 5.5 x 10-5 m; uridine triphosphate, 2 x 10-4 m; pyrophosphate, 8.4 x 10-5 m; and uridine diphosphate glucose, 6 x 10-5 m. Inorganic phosphate inhibits the enzyme competitively with pyrophosphate at a Ki of 4 x10-3 m and uridine diphosphate competitively with uridine diphosphate glucose at a Ki of 1.5 x 10-4 m.