Because of the role that thymidylate synthase (TS) and deoxycytidylate deaminase (DD) play in providing nucleotide products for DNA synthesis, they have become prime targets for chemotherapy. To increase their susceptibility to various inhibitors it is essential to understand the mechanism of action of these enzymes and the manner in which they interact in situ to form thymidylate (dTMP). One approach to limiting dTMP formation and to possibly decrease the toxicity of 5-fluorouracil and 5- fluorodeoxyuridine, is to use a deaminase inhibitor such as pyrimidin-2- one 2'-deoxyriboside, which would reduce the amount of deoxyuridylate available-to compete with 5-fluorodeoxyuridylate for TS. While inhibitors of TS and DD are known, more effective ones can be developed through the technique of rational design. For the latter approach to be successful a three-dimensional analysis of the enzymes is necessary, which has proven to be somewhat successful for TS, but further refinements are necessary. This approach can now be undertaken with DD as we have successfully cloned and amplified both the T4-phage and human enzymes. X-ray studies of the former deaminase are underway and are planned also for the latter enzyme. Studies on the regulation of synthesis of these enzyme at their transcriptional and translational levels are planned, as well as, their influence on cell division. Since we have shown recently in animal cells, that TS is present in the nucleus, its potential interaction with oncogenes and cyclins will be explored, as will the nature of the process by which this enzyme is transported to the nucleus. An in depth analysis of how various structural components affect enzyme activity will be examined by site-specific mutagenesis, differential microcalorimetry and circular dichroism, particularly in the presence of various folate analogues for TS and the allosteric regulators of DD. In the former case at least, we have found TS to be markedly stabilized by folate analogues. It is also planned to isolate a heat stable TS to determine what structural elements contribute to its stability. The role of Zn in the active site of DD will be examined by site-specific mutagenesis and by metal ion replacement studies. The role of the second zinc in T4-phage DD will also be studied by mutagenesis, as will its potential role in stabilizing a purported replication complex of enzymes in phage-infected cells. The location of the binding sites for the substrate of DD, as well as, its allosteric regulators will be explored. And finally, the role of DD as a diagnostic reagent for cancer and other diseases will be examined in sera enzymically, or by interaction with its antibody.
NIH ExPorter
NIH ExPorter