Thymidylate synthase is the target of a number of drugs with real or potential use in cancer chemotherapy. We intend to elucidate details of the mechanism of this enzyme, as well as the mechanism of action of analogs of 2'-deoxyuridylate and the folate cofactor which are potent inhibitors of this enzyme. Where applicable, we will atempt to apply our findings to study and optimize the efficacy of such inhibitors in tissue culture cells, with the anticipation that such studies will be relevant to in vivo systems. We shall attempt to obtain in crystals of the L.casei enzyme which are suitable for X-ray crystallography, and to obtain human tissue culture cell lines which overproduce this enzyme. Should the latter be successful, it would be possible to ascertain whether the human enzyme shares important mechanistic, inhibitory and structural properties with the better understood bacterial thymidylate synthases. We have formulated a hypothesis that the mechanism of thymidylate synthase may serve as a paradigm for many enzymes involved in modifications of the pyrimidine heterocycle. We believe that nucleophilic attack at the 6-position of the pyrimidine heterocycle to form transient 5, 6-dihydro-pyrimidine intermediates is a common feature of many enzymes which modify the 4- and 5-positions of the heterocycle and cleave the glycosidic bond of pyrimidine nucleosides/nucleotides. Using methodologies resulting from studies of thymidylate synthase, we shall test this hypothesis in studies of dCMP hydroxymenthylase, DNA(cytosine) methylases, Cyd and dCMP deaminases, Urd and dThd phosphorylases and PsiUrd synthases. We shall determine the mechanisms of (a) inhibition of DNA(cytosine) methylases by DNA containing 5-azacytosine and 5-fluorocytosine, and (b) inhibition of tRNA PsiUrd synthase by tRNA containing 5-fluorouracil. We believe that when contained in nucleic acids, these analogs form covalent adducts with the aforementioned enzymes they inhibit and are, in fact, mechanism-based inhibitors.
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