title: Purification and properties of the dihydrofolate synthetase from pea seedlings.
abstract: Dihydrofolate synthetase [EC 6.3.2.12] 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, Sephades G-200 gel filtration, and hydroxylapatite column chromatography. The enzyme preparation obtained was confirmed ultracentrifugally to be in the homogeneous state. The sedimentation coefficient of this enzyme was calculated as 3.9S. 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 Mn 2+) 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 X 10 MINUS 6, 1.5 X 10 MINUS 3, 1.0 X 10 MINUS 4, 1.1 X 10 MINUS 3 AND 6.3 X 10 MINUS 5 M, respectively. The enzymatic reaction was inhibited by the addition of ADP, but not by AMP. This suggests that the product fromATPin the reaction is composed of ADP PLUS Pi. Thus, it is proposed that this enzyme catalyzed the following reaction: (see article).
mesh_terms: D000227:Adenine Nucleotides / Q000494:pharmacology; D000645:Ammonium Sulfate; D002413:Cations, Divalent; D002414:Cations, Monovalent; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005971:Glutamates; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000201:enzymology*; D010453:Peptide Synthases / Q000302:isolation & purification* / Q000378:metabolism; D063245:Plant Development; D010944:Plants / Q000201:enzymology*; D011622:Pterins
chemical_list: D000227:Adenine Nucleotides; D002413:Cations, Divalent; D002414:Cations, Monovalent; D005971:Glutamates; D011622:Pterins; D010453:Peptide Synthases; D000645:Ammonium Sulfate