DOMAIN-STRUCTURE AND FUNCTION OF 10-FORMYLTETRAHYDROFOLATE DEHYDROGENASE

10-Formyltetrahydrofolate dehydrogenase catalyzes the NADP(+)-dependen t oxidation of 10-formyltetrahydrofolate to CO2 and tetrahydrofolate. Previous studies have shown that the enzyme binds the physiological pe ntaglutamate form of tetrahydrofolate product so tightly that it remai ns bound during size exclusion chromatography (Cook, R. J., and Wagner , C. (1982) Biochemistry 21, 4427-4434). In addition to the dehydrogen ase activity, the enzyme from rat liver has been reported to exhibit b oth 10-formyltetrahydrofolate hydrolase and aldehyde dehydrogenase act ivities (Cook, R. J., Lloyd, R. S., and Wagner, C. (1991) J. Biol. Che m. 266, 4965-4973). We have purified the enzyme from rabbit liver and found that it catalyzes the same three reactions with similar kinetic constants and that it is a 99-kDa homotetramer, as reported previously for the rat and pig enzymes. Previous studies have suggested that the enzyme is composed of three domains and has separate folate binding s ites for the dehydrogenase and hydrolase activities. We have investiga ted the domain structure of the rabbit enzyme. Differential scanning c alorimetry reveals two thermal transitions, indicating the presence of two independently folded domains. The pentaglutamate form of tetrahyd rofolate and NADP(+) each stabilize one of the thermal transitions, sh owing that these ligands bind to separate domains. Limited proteolytic digestions by several proteases cleave the enzyme in a linker region between the two domains. After proteolytic cleavage, the domains no lo nger remain associated and do not catalyze the 10-formyltetrahydrofola te dehydrogenase reaction. Isolation and characterization of the intac t domains revealed that the N-terminal domain only catalyzes the NADP( +)-independent 10-formyltetrahydrofolate hydrolase activity and the C- terminal domain only catalyzes the NADP(+)-dependent aldehyde dehydrog enase activity. The kinetic constants of these isolated domains are si milar to those of the intact enzyme. Binding studies on the native enz yme using fluorescence and isothermal titration calorimetry indicated that the enzyme binds one molecule of tetrahydrofolate and two molecul es of NADP(+) per tetramer. Dissociation constants for both ligands we re also determined by these methods.