TY - JOUR T1 - Fluorinated Pyrimidines XXVI. Mammalian Thymidylate Synthetase: Its Mechanism of Action and Inhibition by Fluorinated Nucleotides JF - Molecular Pharmacology JO - Mol Pharmacol SP - 14 LP - 30 VL - 1 IS - 1 AU - PHILIP REYES AU - CHARLES HEIDELBERGER Y1 - 1965/07/01 UR - http://molpharm.aspetjournals.org/content/1/1/14.abstract N2 - Thymidylate synthetase from Ehrlich ascites carcinomna cells has been assayed by a spectrophotometric method, and several new properties of this enzyme have been discovered. Thymidylate produces a weak noncompetitive product inhibition and mercaptoethanol stimulates the enzyme activity, whereas formaldehyde must be present within a specific concentration range if maximal enzyme activity is to occur. In addition, the results of initial velocity and product-inhibition kinetic studies as well as those of experiments designed to study the kinetics of the inhibition produced by 5-fluoro-2'-deoxyuridine 5'-monophosphate and 5-trifluoromethyl-2'-deoxyuridine 5'-monophosphate made it possible to elucidate the mechanism of action of thymidylate synthetase. The mechanism is ordered and sequential, such that 5,10-methylenetetrahydrofolate interacts with the enzyme before deoxyuridylate, and thymidylate leaves before dihydrofolate. Furthermore, a mechanism without central complexes has been eliminated. 5-Trifluoromethyl-2'-deoxyuridine 5'-monophosphate, unlike 5-fluoro-2'-deoxyuridine 5'-monophosphate, exhibits the ability to combine slowly with the enzyme in an irreversible fashion. ACKNOWLEDGMENT The authors would like to thank Professor W. W. Cleland for several valuable and helpful discussions during the course of this investigation. This work was supported in part by grants CRTY-5002 and CA-07175 from the National Cancer Institute, United States Public Health Service, Bethesda, Maryland. ER -