This proposal involves a multi-disciplinary research effort directed at structure/function/inhibition studies of enzymes in pyrimidine nucleotide and nucleic metabolism which share common mechanistic themes. These enzymes include thymidylate synthase, DNA-cytosine methyltransferase, tRNA-methyltransferase, and deoxycytidylate hydroxymethylase. Our objective are aimed at obtaining fundamental biochemical information, with the belief that such knowledge will provide insight to exploit what is found to assist in controlling disease. These studies are particularly relevant to the understanding of anticancer agents such as fluorouracil and 5,8- dideazafolates, and in the design of new chemotherapeutic agents. We will continue and extend our studies on the structure, function and inhibition of the enzyme thymidylate synthase. These include studies of mechanism-based inhibitors, detailed kinetic studies of inhibitors such as 5-fluoro-2'-deoxyuridylate, and mutagenesis; they also include computer-assisted molecular modelling and structural investigations. Most of our initial studies will use the enzyme from L. casei. We will also complete studies on the heterologous expression of the human enzyme and, when appropriate, redirect efforts towards this important enzyme. We shall continue investigations on the DNA cytosine methyltransferases which are directed at understanding the mechanism of these enzymes, as well as aspects of DNA-enzyme interactions. We shall continue studies on the tRNA methyltransferase. Here, we shall develop analogous high-expression systems, investigate the catalytic mechanism of this enzyme, and investigate studies on recognition of tRNA by systems, investigate the catalytic mechanism of this enzyme, and investigate studies on recognition of tRNA by this enzyme. finally, we shall attempt to identify the active site nucleophile of deoxycytidylate hydroxymethylase.
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