Inhibition of thymidylate synthetase by showdomycin and its 5′-phosphate

Thymidylate synthetase of dichloromethotrexate resistant Lactobacilluscasei was found to be rapidly and irreversibly inactivated by showdomycin (3-β-D-ribofuranosylmaleimide). Prior treatment of the enzyme with hydroxyethyldisulfide prevented the inhibitory effect of showdomycin. The enzyme could also be protected by its substrate, deoxyuridylate, against inactivation by the antibiotic. Reduction of the 3, 4-double bond by catalytic hydrogenation destroyed the enzyme inhibitory activity of showdomycin. Phosphorylation of its 5′-hydroxyl group greatly enhanced the inhibitory potency of the antibiotic, presumably by conferring substrate-like specificity. These results suggest that the maleimide moiety of showdomycin alkylates a reactive sulfhydryl group at the active site of thymidylate synthetase, and are consistent with a sulfhydryl addition-elimination mechanism of the enzyme catalyzed reaction.