Purification and characterization of Thymidine 5-monophosphate kinase from Escherichia coli B.

Thymidylate kinase was purified about SOOO-fold from Eschenkhia coti B. The final steps in the purification procedure utilized preparative disc gel electrophoresis, and the most highly purified preparation appeared to be homogeneous with respect to particle size, sedimentation velocity, and electrophoretic mobility. The enzyme phosphorylated deoxythymidine monophosphate, 5-iodo-2’-deoxyuridylic acid, and deoxyuridine monophosphate, in that order of activity, with ATP as the phosphate donor. Several other nucleoside triphosphates could serve less effectively as phosphate donors in the reaction. Magnesium ion, which could be replaced partially by either manganese or cobalt, was an absolute requirement. 5-Iodo-2’-deoxyuridylic acid and dUMP were competitive inhibitors of the phosphorylation of dTMP. ATP, deoxycytidine triphosphate, or deoxythymidine triphosphate in excess of the stoichiometrically equivalent concentration of Mg++ strongly inhibited the reaction. The inhibitory effect of ATP and dCTP was abolished when the Mg+concentration was increased to equal the total nucleoside triphosphate concentration. dTTP inhibited the reaction about 30% even when Mg+f was present in excess of the total concentration of triphosphates.