Molecular Pharmacology, Vol 6, 621-630, Copyright © 1970 by the American Society for Pharmacology and Experimental Therapeutics

Enzymatic Studies Relating to the Mode of Action of 5-Mercapto-2'-deoxyuridine

¹ Departments of Biochemical Pharmacology and Medicinal Chemistry, Faculty of Health Sciences, State University of New York at Buffalo, Buffalo, New York 14214

Kinetic studies of the effects of 5-mercapto-2'-deoxyuridine (MUdR) and some of its derivatives on the thymidine kinase and thymidylate synthetase of Escherichia coli B are presented. It was found that MUdR and its S-methyl derivative are phosphorylated by thymidine kinase, while other derivatives, including the disulfide, are not. In crude extracts of E. coil B, thymidylate synthetase was inhibited by MUdR (but not the S-methyl derivative) in the presence of ATP only. MUdR 5'-monophosphate (MUdRP) was prepared by enzymatic phosphorylation of MUdR and found to be a potent inhibitor of purified E. coil B thymidylate synthetase (K_i = 4.0 x 10^-8 M; K_i/K_m = 0.0085), competitive with the substrate, deoxyuridylate. The "anomalous" strong binding of MUdRP to this enzyme is discussed.

The results of these studies are consistent with the hypothesis that the mode of action of MUdR involves the metabolic activation of the analogue via phosphorylation by thymidine kinase and subsequent inhibition of thymidylate synthetase by the active form, MUdRP, resulting in the inhibition of DNA synthesis.

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ACKNOWLEDGMENTS The technical assistance of Mrs. M. Hsiao and Mr. M. Anderson in part of this work is appreciated.