In the cytosol there are three major anabolic pathways which utilize folate requiring enzymes. These include the synthesis of thymidylate, the synthesis of purines, and the synthesis of methionine and other methylated compounds. although most research has been performed with the monoglutamate form of the coenzyme (H4PteGlu), in the cell an enzyme called folyl polyglutatamate synthetase catalyzes the addition of glutamate residues linking each successive Glu as an amide through the gamma-carboxyl group. In most cells the coenzyme exists as pools of polyglutamates containing between 5 and 7 glutamate residues (H4PteGlu5- 7). Nobel Laureates Hitchings and Elion recognized that inhibitors of folate enzymes could be effective anticancer agents by blocking the reactions required for cell division. In 1943 the average life span of a child with acute leukemia was 3 months. today there is better than a 75% cure- rate for these children. This is the result of the use of antifolate drugs like methotrexate and 5-fluorouracil. Antifolate drugs are now used routinely in the treatment of many cancers, diseases caused by trypanasome infections, and as antibiotics (sulfa drugs). Antifolates which inhibit key folate enzymes, like serine hydroxymethyltransferase, are still not available. The design of possible good inhibitors of folate enzymes can be enhanced by determining the mechanism of folate enzymes and describing the probable structure of the transition-state. The design of transition-state inhibitors has proved an effective method for bringing some rational thought to drug design. The three broad goals of the proposed research are to continue to determine; (1) the mechanism and function of several enzymes in one- carbon metabolism, (2) the role of the polyglutamate moiety of tetrahydropyterolypolyglutamates (H4PteGlun), and (3) the chemistry and metabolic function of 5-formylH4PteGlun.
Specific Aim 1 : Determine the metabolic role of 5-CHO-H4PteGlun. Two possible functions will be explored: 1) as a regulator of one-carbon metabolism, 2) as a storage form of one-carbon units.
Specific aim 2 : Determine the structure and mechanism of 5,10-methenyl tetrahydrofolate synthetase and 10-formyl tetrahydrofolate dehydrogenase. These two enzymes are believed to play critical roles in regulating one-carbon metabolism, yet little is known about their structure and mechanism.
Specific Aim 3 : Determine the role of active site residues in the mechanism of serine hydroxymethyltransferase.
Specific Aim 4 : Determine the role of H4PteGlun polyglutamates and pyridoxal-P in one-carbon metabolism. Two possible functions will be explored: 1) the polyglutamate moiety as a structural requirement in the channeling of the coenzyme between folate requiring enzymes, and 2) as chaperones in protein folding.
Showing the most recent 10 out of 46 publications
