. This revised proposal focuses on mechanistic and structural studies on thymidylate synthase/dihydrofolate reductase (TS-DHFR) . These two enzymes are crucial for DNA synthesis and one-carbon transfers. In many protozoan parasties, these two catalytic activities are located on a single polypeptide chain to form the bifunctional TS- DHFR. In mammals, these enzymes are separate and monofunctional. A considerable amount of mechanistic information is available for the human monofunctional TS and DHFR, since each enzyme has been successfully targeted with the anticancer drugs, 5-fluorouracil and methotrexate, respectively. Earlier work as well as preliminary transient kinetic studies from the PI's laboratory indicate substantial mechanistic differences in the bifunctional parasitic enzyme and the monofunctional human enzymes. The ultimate goal of the proposed research is to take advantage of the differences between the bifunctional parasitic enzyme and the human enzymes to develop novel drugs for the treatment of parasitic diseases. To accomplish this goal, the following specific aims are proposed: 1) Elucidate the molecular mechanism involved in enzyme catalysis and substrate channeling for the bifunctional TS-DHFR from Toxoplasma gondii, 2) Use site-directed mutagenesis as a tool for mechanistic and structural studies with the Leishmania TS-DHFR and the Tosoplasma TS-DHFR, and 3) Use combinatorial library screening in conjunction with computer modeling to identify compounds that interfere with substrate channeling.
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