Properties and inhibition of thymidylate synthetase in Drosophila melanogaster

A quantitative comparison of the incorporation of methyl-3H-thymidine and 6-3H-deoxyuridine into the DNA of Drosophila melanogaster in the presence and in the absence of 5-fluorouracil indicated that 5-fluorouracil inhibits the reaction converting dUMP to dTMP catalysed by thymidylate synthetase (methylenetetrahydrofolate:dUrd-5'-P C-methyltransferase, E.C. 2.1.1.b). The enzyme exhibits maximal activity at pH 7[middle dot]5 to 8[middle dot]0 and is protected from heat inactivation by deoxyuridine monophosphate. The addition of thiol compounds to the homogenization buffer results in the enhancement of synthetase activity. The Km values for deoxyuridine monophosphate and 5,10-methylenetetrahydrofolate are 6[middle dot]8 x 10-6 M and 8[middle dot]3 x 10-5 M, respectively. Fluorodeoxyuridine monophosphate, trifluoromethyldeoxyuridine monophosphate, and methotrexate are inhibitors of the enzyme. 5-Bromodeoxyuridine and 5-iododeoxyuridine have no inhibitory effect. The results support the contention that, under conditions which induce morphological lesions in Drosophila, fluorinated pyrimidines and methotrexate inhibit the de novo synthesis of thymidylate whereas thymidine analogues function in some other manner.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22328/1/0000773.pd