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Studies on Dihydrofolic Reductase

I. PURIFICATION AND PROPERTIES OF DIHYDROFOLIC REDUCTASE FROM CHICKEN LIVER

From the ¹ From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014

Dihydrofolic reductase has been purified approximately 8000-fold from chicken liver and appears to be essentially homogeneous by the criterion of aminopterin inhibition. The observed turnover number in the range of 300 (dihydrofolate at pH 7.4) agrees with various calculated values. The titration with aminopterin also confirmed the remarkably low molecular weight (23,000) for a pyridine nucleotide enzyme and suggests one binding site per molecule.

The most effective stabilizing agent for the enzyme was triphosphopyridine nucleotide or its reduced form. The binding of TPN is reflected in its ability to act as a potent competitive inhibitor of the reductase reaction (K_i = 2.4 x 10^-6 m).

With dihydrofolate and TPNH, the purified enzyme exhibits two pH optima, 4 and 7.4. At the neutral pH optimum, the reaction is specific for these two substrates. In the acid pH range, DPNH may be utilized as the reducing agent with dihydrofolate (pH optimum, 5.5), and folate exhibits significant substrate activity with TPNH (pH optimum, 3.8). Also in the acid range, citrate is an inhibitor of the reduction of dihydrofolate, but not folate.

Kinetic analysis of the interaction of dihydrofolate with the enzyme and TPNH yielded a sigmoid curve with an estimated K_m of approximately 1.5 x 10^-7 m at pH 7.4. Although V_max is 3 to 4 times higher at pH 4.5 than pH 7.4, the K_m appears to be the same at both pH ranges. The interaction of folate with the enzyme and TPNH yields "normal" kinetics with a K_m of 6.7 x 10^-6 (citrate buffer). The interaction of TPNH with the enzyme and dihydrofolate reveals "normal" kinetics at pH 7.4 and "sigmoid" kinetics at pH 4 with either dihydrofolate or folate. However, the apparent K_m appears to be the same (1.6 x 10^-6) with either substrate and at either pH.

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