The ATP- and Mg2+-dependent conversion of 5-formyltetrahydrofolate to the 5,10-methenyl derivative, catalyzed by 5,10-methenyltetrahydrofolate synthetase (EC 6.3.3.2) is one of only two reactions in tetrahydrofolate-mediated one-carbon metabolism which utilize 5-formyltetrahydrofolate as the normal substrate. This reaction is of importance in cancer chemotherapy, since it may provide the basis for """"""""leucovorin-rescue"""""""" from the deleterious effects of high-dose Methotrexate regimens. The reaction is representative of a special class of ATP-dependent enzymes catalyzing the formation of an amidine from an amide (in this case both the amine and amide reside on the same molecule resulting in a cyclic amidinium). A detailed investigation into the kinetic and chemical mechanism of the reaction catalyzed by the enzyme purified to homogeneity from Lactobacillus casei will thus provide significant insight into the mechanism of enzymic catalysis in this intriguing reaction. Particular emphasis will be applied to elucidation of the detailed stereochemistry of the ATP hydrolysis step, since no information currently exists for this type of ATP-dependent reaction. The precise mechanism for coupling of the energy of ATP hydrolysis to the cyclization reaction is uncertain, since enzyme-catalyzed cyclization of the isosteric 5-formimino derivative shows no requirement for ATP or M2+. Characterization of the stereochemical requirements for binding and catalysis of the M2+-ATP chelate complex will be determined using nucleoside 5'-phosphorothioates and substitution-inert chelate complexes of Cr3+-ATP and ADP. The chromophoric properties of the H4folate substrate and product will be utilized to conduct detailed kinetic studies of the reaction mechanism, and hopefully obtain spectrophotometric evidence for formation of a phosphorylated intermediate. Using the knowledge gained from the L. casei enzyme, a comparison will be made with the mammalian enzyme isolated from rabbit liver. Key experiments will be conducted to determine the structural and mechanistic basis for the apparent differences in kinetic mechanism, affinity for the nucleoside 5'-triphosphate substrate, and affinity for inhibitory folate derivatives.
