Uridine Diphosphate Sugar Hydrolase: PURIFICATION OF ENZYME AND PROTEIN INHIBITOR

Abstract

The partial purification of a uridine diphosphate sugar hydrolase from Escherichia coli ATCC 12793 and a specific protein inhibitor of this enzyme are described. The products of the hydrolysis are uridine, inorganic phosphate, and sugar 1-phosphate. The enzyme appears to be identical with the 5'-nucleotidase in this organism. In addition, these cells contain a specific adenosine diphosphate-glucose pyrophosphatase. The 5'-nucleotidase is relatively nonspecific, but the uridine diphosphate sugar hydrolase activity cleaves only uridine nucleotides at a significant rate. Other nucleoside diphosphate sugars containing adenine, guanine, or cytosine as the base are cleaved at less than 5% of the rate of the uridine nucleotides, although the apparent affinity of these nucleotides for the enzyme is the same as that of the uridine nucleotides. Both enzymatic activities show an absolute requirement for the addition of a divalent cation. At pH 8.0 in Tris-Cl buffer, this requirement can be satisfied in order of decreasing affinity by Co++, Mn++, and Mg++, while at pH 5.1 in acetate buffer only Co++ and Mn++ are effective. These same metals protect the enzyme against heat inactivation. Similar, if not identical, enzymes are present in several other strains of E. coli, and a similar enzyme is also present in Salmonella weslaco. All of these enzymes are released into the medium when cells are converted to spheroplasts, but the inhibitor remains in the spheroplast. In Salmonella typhimurium LT-2, a soluble adenosine diphosphate-glucose pyrophosphatase and a particle-bound nucleoside diphosphate sugar pyrophosphatase of broader specificity which is not associated with 5'-nucleotidase activity are present. Some of the properties and possible functions of these enzymes are discussed.