ENZYMATIC SYNTHESIS OF HYDROXYMETHYLDIHYDROPTERIDINE PYROPHOSPHATE AND DIHYDROFOLATE

The enzymes involved in the reactions leading to dihydrofolate from extracts of Lactobacillus plantarum were resolved into two fractions by DEAE-cellulose chromatography. Fraction I catalyzed the pyrophosphorylation of 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine (hydroxymethyldihydropteridine) to 2-amino-4-hydroxy-6-pyrophosphorylmethyl-7,8-dihydropteridine (hydroxymethyldihydropteridine pyrophosphate). Fraction II coupled the latter pteridine with p-aminobenzoylglutamate or p-aminobenzoate to give dihydrofolate or dihydropteroate. These conclusions were based on experimental findings which show that the reaction catalyzed by fraction I requires adenosine triphosphate and hydroxymethyldihydropteridine. Furthermore, the isolated product of this reaction had a spectrum similar to a dihydropteridine, contained two phosphate moieties, incorporated 32P indicating the transfer of the P-32P residue from adenosine triphosphate-γ-32P, and when chromatographed with carrier hydroxymethyldihydropteridine pyrophosphate gave the expected specific activity in fractions collected from a DEAE-cellulose column. Fraction II utilized the product of the above enzymatic reaction or synthetic hydroxymethyldihydropteridine pyrophosphate to synthesize dihydrofolate or dihydropteroate with p-aminobenzoylglutamic acid or p-aminobenzoic acid, respectively. A sigmoidal kinetic response was observed with each cosubstrate. This fraction was completely inactive in a system containing hydroxymethyldihydropteridine, adenosine triphosphate, and p-aminobenzoylglutamic acid or p-aminobenzoic acid. We suggest that the enzymes of fractions I and II be named hydroxymethyldihydropteridine pyrophosphokinase and dihydropteroate or dihydrofolate synthetase, respectively. © 1969, American Chemical Society. All rights reserved.