The objective of this proposal is the use of NMR, mechanistic strategies, and immunochemical approaches to study thymidylate synthase which catalyses the 5, 10-methylenetetrahydrofolate- dependent reductive methylation of deoxyuridylate to form thymidylate. Its strategic role in thymidylate biosynthesis has make the latter enzyme an attractive target for selected pyrimidine deoxynucleotides and antifolates which serve as drugs in the treatment and management of certain cancers. In view of the expected x-ray crystallographic determination of the three- dimensional structure of Lactobacillus casei thymidylate synthase, we propose the cloning of the L. casei gene for this enzyme and the investigation of catalysis, binding, and structural aspects of the enzyme via site-directed mutagenesis. We also propose to isolate, purify, and characterize thymidylate synthase from fluorodeoxyuridine-resistant HEp-2 cells (human laryngeal carcinoma cell line) and compare its properties with those of the L. casei enzyme. Immunological studies are directed at creating enzyme-linked immunoassays of thymidylate synthase and its modified forms and at studying structure-function relationships in these proteins. Characterization of mechanistic steps in the formation of binary and ternary complexes is planned through the use of a variety of NMR and biochemical approaches.
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