Folic acid is necessary for the synthesis of metabolites that are required for cell growth. For this reason, the folate metabolic pathways have been targets for therapies aimed at blocking cell division using antifolates. Cellular uptake of antifolate monoglutamate occurs via specific transporters. Intracellular classical antifolates are converted to poly-gamma-glutamates by polyglutamate synthetase. The poly-gamma-glutamate derivatives cannot cross the cell membrane, concentrate intracellularly, and can be better substrates or inhibitors for the folate dependent enzymes. Formation of antifolyl poly-gamma-glutamates and cellular retention are countered by removal of the poly-gamma-glutamate tail catalyzed by a poorly understood enzyme, gamma-glutamyl hydrolase. Cellular folate homeostasis and the efficacy of certain antifolates such as methotrexate are determined by the balance between the activities of these two enzymes. The goal of the proposed research is to define the molecular details of the mechanism of action of human gamma-glutamyl hydrolase (hGH) and to determine the structural features that contributed to the specific mode of action of this enzyme. The mechanism of hGH will be investigated by identifying the active site residues using site directed mutagenesis to test a molecular model for the catalytic fold of the enzyme. Amino acids, which bind the poly-gamma-glutamate chain of substrates will be identified by site directed mutagenesis. The three dimensional structure of the enzyme will be determined by X-ray crystallography. Transfection with cDNA or antisense constructs will be used to alter hGH expression in cancer cells to establish the role of this enzyme in antifolate and fluorouracil action. These studies will determine if GH is a potential site for modulation of current therapy with these drugs. The characterization of an identified cellular inhibitor of human gamma-glutamyl hydrolase will also be carried out. The specific inhibition of hGH is necessary to determine its role in folate homeostasis and antifolate action. The results of the proposed studies may lead to the development of specific inhibitors of human gamma-glutamyl hydrolase, which could be used in conjunction with established antifolates or fluoropyrimidines in the treatment of human cancers.
