Dihydropteroate synthetase was purified from extracts of one day-old pea seedlings by chromatographic methods on DEAE-cellulose, DEAE-Sephadex A-50 and Sephadex G-200. The final enzyme preparation was homogeneous in chromatographic and ultracentrifugal analyses. The purified enzyme utilized 2-amino-4-hydroxy-6-hydroxy-methyldihydropteridine as substrate in the presence of both ATP and Mg²⁺, as did its pyrophosphate ester in the presence of Mg²⁺, to produce dihydropteroic acid by coupling with p-aminobenzoic acid. The Km value for pyrophosphorylmethyldihydropteridine was 1.14×10^-5M. The enzyme was specific for the amino group at the para-position in benzoic acid, since the reaction was not influenced by the addition of ortho- or meta-aminobenzoic acid. The Km value for p-aminobenzoic acid was 1.11×10^-6M.
Enzymatic formation of dihydropteroic acid was competitively inhibited by several sulfonamides. Inhibition levels of the various sulfonamides corresponded relatively with the growth inhibition levels by these compounds for Escherichia coli.