This proposal involves nmr and mechanistic studies of thymidylate synthetase which catalyzes the 5,10-methylenetetrahydrofolate-dependent reductive methylation of deoxyuridylate to form thymidylate and 7,8-dihydrofolate. Its strategic role in the chief de novo pathway for thymidylate biosynthesis has made this enzyme an attractive target for the action of substituted pyrimidines, such as 5-fluorouracil, which serve as drugs in the treatment and management of certain cancers. In this proposal we develop a series of nmr and biochemical techniques which are then applied to answer important questions concerning the interaction of the inhibitor, 5-fluorodeoxyuridylate, with the enzyme. We seek to determine the stereochemical consequences and chemical dynamics associated with FdUMP binding along with the effect of chemical modification on inhibitor interaction. We also want to know if FdUMP binding at one site on the enzymic dimer affects interaction at the other site. We then intend to pose similar questions with respect to the interaction of the natural substrate, dUMP, and the natural product, dTMP, with the enzyme. The resulting data will be used to determine the validity of mechanistic schemes proposed for nucleotide binding to the enzyme. We will also examine the mechanistic consequences of a covalent enzyme-dTMP complex, and we will employ 19F nmr and certain biochemical tests as a means of comparing certain mechanistic features of the Lactobacillus casei enzyme with those of a mammalian thymidylate synthetase. We will initiate studies of the interaction of folate ligands in binary and ternary complexes and will perform nmr studies to determine the site linkage of the methylene group in the inhibitory ternary complex. The nature of the active site cysteinyl residues will be probed by crosslinking studies and chemical modification with reporter groups. Our initial studies on tyrosine modification are extended in an effort to determine the role of tyrosine in enzyme activity.
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