Because of the role that thymidylate synthase (TS) and deoxycytidylate deaminase (DD) play in providing, as well as regulating the supply of nucleotide products for DNA synthesis, these enzymes are targets for chemotherapy. We have studied the two enzymes extensively over a period of 27 years and have reached a point where we can provide significant information on the topography of both enzymes which might be exploited for the purpose of developing even more effective chemotherapeutic agents. Thus, in the case of TS, the binding sites of the folate substrate and its analogues methotrexate and 10-propargyl-5,8- dideazafolate will be determined, as well as that of photofixed deoxyuridine 5'-monophosphate and its 5-azido derivative. In addition site-directed mutagenesis will be employed to evaluate those regions of the protein which influence enzyme activity. This technique will also be used to study the mechanism of splicing associated with the intron-containing td gene encoding T4-phage TS. Methods will be developed to isolate and to amplify the expression of the human TS gene for purposes of x-ray crystallography. Cell culture studies will be conducted to examine the mechanism by which CB3717 affects its inhibitory potential. In the case of DD, similar studies on infrastructure probing will be conducted particularly with reference to its substrate and allosteric binding sites. These studies should be greatly aided by site-directed mutagenesis. Isolation and sequencing of the human DD gene is planned, as well as its amplification by a high expression vector, which will provide sufficient amount of protein for x-ray crystallographic studies. Location of the intracellular compartment of the enzyme will be established by immuno-electron microscopy. Eventually, it is hoped to examine the possibility that TS and DD are elevated in the sera of tumor containing animals, which would portend their use as diagnostic tools.
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