The characteristics and significance of sulfonamides as substrates for Escherichia coli dihydropteroate synthase.

Sulfonamides are known to compete with p-aminobenzoic acid for dihydropteroate synthase. Others have reported that some sulfonamides are alternate substrates, but the significance of these observations to the antimicrobial action of sulfonamides has not been studied. We have shown that sulfanilamide, sulfathiazole, and sulfamethoxazole are efficient alternate substrates for this reaction, with apparent K,,, values equivalent to their Ki values as competitive inhibitors. The products synthesized from the sulfonamides in uitro were chromatographically similar to chemically prepared dihydropterin-sulfonamides. A culture of Escherichiu coli B converted 29% of 0.625 pM [35S]sulfamethoxazole to a product which was identified as dihydropterin-sulfamethoxazole. Greater than 99% of the product was found in the medium and the cellular concentration of radiolabel was 52 pM. This lack of accumulation was consistent with our finding that sulfonamides diffuse into E. coli and that the active transport of [35S]sulfanilamide could not be demonstrated. The growth rate of E. coli B was not inhibited by 2 pM of chemically synthesized dihydropterin-sulfamethoxazole. No significant inhibition of thymidylate synthase, N’,N”-methylenetetrahydrofolate dehydrogenase, N’,N”-methenyltetrahydrofolate cyclohydrolase, or dihydrofolate reductase was found with the aromatic and dihydropterin-sulfonamides. High concentrations (50 to 150 pM) of some of the compounds were inhibitory to GTP cyclohydrolase, hydroxymethyldihydropterin pyrophosphokinase, and serine hydroxymethyltransferase. The dihydropterin-sulfonamides were product inhibitors of dihydropteroate synthase, and were inhibitors of dihydrofolate synthetase. However, to obtain substantial inhibition of these enzymes by the dihydropterin-sulfonamides in duo, higher concentrations of these compounds are required than those which are attainable intracellularly. These data show that sulfonamides are effective alternate substrates for E. coli dihydropteroate synthase, but that the dihydropterin-sulfonamide products formed do not contribute significantly to the growth inhibition by sulfonamides.