Abstract

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.