This proposal outlines a research effort centered on the two important folate-requiring enzymes in de novo purine biosynthesis, 5,10-methenyltetrahydrofolate: 2-amino-N-ribosylacetamide-5'-phosphate transformylase and 10-formyltetrahydrofolate: 5-amino-1-ribosyl-4-imidazolecarboxamide-5'-phosphate transformylase (both from liver). Experiments will be conducted that are designed to 1) obtain both enzymes in a homogeneous state; 2) characterize the physical and structural properties with respect to protein composition and associated activities; 3) delineate the kinetic and chemical events associated with the mechanism of one-carbon unit transfer, particularly the sequence of appearance and structure of kinetically competent intermediates; and 4) demonstrate and then quantitate inhibitory interactions between the two antifolate analogs, methotrexate and tetrahydrohomofolate, and one and/or obth of the transformylases in order to substantiate a multiple site of action hypothesis for the two chemotherapeutic agents. Information gained will be used to design specific agents for treatment of neoplastic disease owing their cytotoxicity to inhibition of purine biosynthesis through interaction with the transformylase enzymes.