Studies on Dihydrofolic Reductase

The activity of chicken liver dihydrofolic reductase, which catalyzes the reduced pyridine nucleotide-dependent reduction of folate or dihydrofolate to tetrahydrofolate, is stimulated approximately 4-fold by 4 m urea and 8-fold by 1.6 m thiourea when TPNH and dihydrofolate serve as substrates. When folic acid and TPNH are the substrates, relatively slight stimulation of activity is observed with concentrations of urea or thiourea less than 1 m . At higher concentrations, these reagents are inhibitory. On the other hand, both urea and thiourea are markedly inhibitory when DPNH and dihydrofolate are used as substrates. With increasing concentrations of urea, the double pH-activity profile (optima at pH 4 and pH 7.4) of the enzyme is shifted to a profile exhibiting a single pH optimum, at approximately 6.5, when TPNH and dihydrofolate serve as substrates. No significant changes are observed when folate and TPNH or dihydrofolate and DPNH are used as substrates. As the concentration of urea or thiourea is increased to the optimum concentration for stimulation, the Vmax increases approximately 4- to 10-fold with respect to TPNH or dihydrofolate, whereas as the apparent Km for dihydrofolate increases approximately 80-fold the Km for TPNH increases only about 4-fold. Under similar conditions, no significant changes in the Km values for folate or DPNH are evident. The enthalpy of activation also increases with increasing concentration of the activating reagents, from +12.6 to +15.4 kcal per mole. At optimum stimulatory concentrations of urea or thiourea, the first order half-life for enzyme activity is markedly decreased (1½ min at 30°). This half-life increases to 22 min in the presence of 0.1 m m TPNH. Significant stabilization of the stimulated enzyme is observed also in the presence of similar concentrations of folate, dihydrofolate, TPN, or 20% glycerol. This loss of enzyme activity in 4 m urea is reversible on dilution, whereas the loss in 1.4 m thiourea appears to be irreversible. These observations are consistant with an activation of dihydrofolic reductase resulting from a conformational change in the enzyme protein induced by the presence of high concentrations of urea or thiourea concomitant with the binding of TPNH.