Methylation of 4-thio-2′-deoxyuridylate by thymidylate synthetase

4-Thio-2′-deoxyuridylate was found to be a substrate for the thymidylate synthetase of a dichloromethotrexate resistant strain of Lactobacillus casei. The enzymic product which contained the carbon derived from formaldehyde, was identified as 4-thiothymidylate by chromatographic analysis. 4-Thio-2′-deoxyuridylate inhibited the methylation of deoxyuridylate with an apparent K_i of 7×10^-5 M, reflecting its lower affinity for the enzyme as compared to the natural substrate. 4-Thio-2′-deoxyuridylate is, so far, the only substrate analog modified in the base which is methylated by thymidylate synthetase.