1975 Volume 21 Issue 1 Pages 7-18
Dihydrofolate synthetase [EC 184.108.40.206] was extracted from the cell particles (mitochondrial fraction) of pea seedlings and purified about 2, 000-fold by ammonium sulfate fraction, DEAE-cellulose column chromatography, Sephadex G-200 gel filtration, and hydroxylapatite column chromatography. The enzyme preparation obtained was con-firmed ultracentrifugally to be in the homogeneous state. The sedimen-tation coefficient of this enzyme was calculated as 3.9 S. The apparent molecular weight of the enzyme was determined to be about 56, 000.
Optimum pH for the reaction was 8.8. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and divalent (Mg2+ or Mn2+) and univalent (K+, NH4+ or Rb+) cations as cofactors. The enzyme was specific for dihydropteroic acid as the substrate. ATP was not replaceable with any other nucleotides. Km values for dihydropteroate, L-glutamate, ATP, Mg2+, and Mn2+ were 1.0×10-6, 1.5×10-3, 1.0×10-4, 1.1×10-3, and 6.3×10-5M, respectively. The enzymatic reaction was inhibited by the addition of ADP, but not by AMP. This suggests that the product from ATP in the reaction is composed of ADP+Pi.
Thus, it is proposed that this enzyme catalyzes the following reaction:
Dihydropteroic acid+L-glutamic acid+ATP Mg2+ (or Mat+)→K+ (or NH4+ or Rb+)