The proposed research involves determining the structure, mechanisms, and regulation of serine hydroxymethyltransferase and the trifunctional enzyme C1-tetrahydrofolate synthase from rabbit liver. A coupled system involving these two enzymes and four catalytic activities converts formate to serine using catalytic amounts of the coenzyme tetrahydropteroyl glutamate. The research is also concerned with the role of the polyglutamate forms of the coenzyme in this system. These two reactions are important to one- carbon metabolism because they are the major source of all one- carbon groups for the biosynthesis of methyl containing compounds, thymidylate, and the purine ring. Control of this pathway is important in the use of such chemotherapeutic drugs as methotrexate and fluorouracil. Specific aims include: determining Km and kcat values for each pteroyl polyglutamate for each reaction in the cycle; determining on and off rate constants by stopped-flow spectrophotometry for each polyglutamate; determining the mechanism of addition and release of substrates and products for each reaction; determining the amino acid sequence of the hinge region which joins two domains in C1-tetrahydrofolate synthase; using monoclonal antibodies to map the active sites of this multifunctional enzyme; and cloning and expressing the individual domains of this multifunctional enzymes. The kinetic properties of the coupled enzyme system will be studied by computer modeling. The results of this study should determine if there is channeling of tetrahydropteroyl polyglutamates between active sites, if the rate limiting step for any of the reactions is the dissociation of products, and if a complex is formed between serine hydroxymethyltransferase and C1-tetrahydrofolate synthase.