16] Pyrimidine nucleoside and deoxynucleoside phosphorylases

Abstract

This chapter describes the purification, assay method, and properties of two enzymes; pyrimidine deoxynucleoside phosphorylase from Escherichia coli; and uridine phosphorylase from Escherichia coli. Purification procedure and properties of these enzymes are mentioned. The specificity studies conclude that every position of the base and sugar of thymidine is recognized by the enzyme; thus, it is impossible to predict whether other analogs may function as substrates or inhibitors, but every part of the molecule is of importance to the enzyme. The enzyme is dependent on a reducing agent for stability. It is inactivated rapidly at pH values below four and above nine; heating at 70° for five minutes results in complete loss of activity. Purification procedure includes first growth of cells and preparation of extracts, then protamine sulfate and ammonium sulfate precipitations, and finally alumina Cy gel fractionation. Preparation of nucleosides and deoxynucleosides involves reaction mixtures containing pyrimidine base and pentose phosphate in 0.005 M Tris plus 0.005 M 2-mercapteethanol, pH 7.5, eventually reach equilibrium when pentoside formation is about 60%.