We propose to synthesize folic acid and its analogs or inhibitors bearing 13C- or 15N-stable isotope labels at specific positions. These compounds will be incubated with dihydrofolate reductase (DHFR) from bacterial sources; any shifts in the 13C- or 15C-NMR signals between bound and unbound compounds will be noted and evaluated for their indication of involvement of the particular labeled atom in binding at the active site region of the enzyme. We hope to elucidate the mechanism of attachment of folic acid and its inhibitors, such as methotrexate, to this key enzyme. Because this enzyme, DHFR, is crucial to the powerful antitumor and antimicrobial activity of folate analogs, a determination of its mechanistic aspects in binary and ternary complexes with coenzyme is vital to the design of better agents. We believe that the proposed research will allow dynamic assessment of intimate detail and constitute an improvement over more classical instrumental measurements or X-ray solid-state predictions.
| Birdsall, B; De Graw, J; Feeney, J et al. (1987) 15N and 1H NMR evidence for multiple conformations of the complex of dihydrofolate reductase with its substrate, folate. FEBS Lett 217:106-10 |
