The Enzymatic Mechanisms for Deoxythymidine Synthesis in Human Leukocytes

In a cell-free system prepared from human leukocytes, synthesis of deoxythymidine is catalyzed by a transfer of deoxyribosyl from a deoxynucleoside to thymine (pyrimidine deoxyribosyltransferase) and by a coupled deoxynucleoside phosphorylase mechanism in which deoxythymidine is synthesized from the deoxyribose-1-P formed after phosphorolytic cleavage of either purine or pyrimidine deoxynucleosides. Both enzymatic mechanisms participate in deoxythymidine synthesis with pyrimidine deoxynucleosides as the deoxyribosyl donor. However, the proportion synthesized by either mechanism is dependent on the concentration of deoxynucleoside. At concentrations above 5 mm the transferase is the predominant enzymatic mechanism. Purine deoxynucleosides, on the other hand, participate in deoxythymidine synthesis only by a coupled deoxynucleoside phosphorylase mechanism. Rates of deoxythymidine synthesis with purine deoxynucleosides as deoxyribosyl donors were approximately one-tenth to one-thirtieth of the rates with pyrimidine deoxynucleosides. The constant ratio of transferase to phosphorylase through a 140-fold purification and during heat inactivation studies suggests that both activities are the function of one protein. In the presence of thymine and both deoxyuridine and deoxyribose-1-P, synthesis of deoxythymidine occurs independently by both enzymatic mechanisms, indicating that there are separate sites for each deoxyribosyl donor.