The Enzymic Synthesis of Dihydropteroate and Dihydrofolate by Plasmodium berghei

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Abstract
SYNOPSIS. Cell‐free extracts of the rodent malaria parasite Plasmodium berghei synthesized dihydropteroate (H2pteroate) and dihydrofolate (H2folate) from 2‐amino‐4‐hydroxy‐6‐hydroxymethyl‐7,8‐dihydropteridine (hydroxymethyldihydropteridine) and p‐aminobenzoate (pAB) or p‐aminobenzoylglutamate (pABG). The reaction was demonstrated also in extracts of Plasmodium gallinaceum, Plasmodium lophurae and Plasmodium knowlesi, by the use of a microbiologic assay method and pABG as cosubstrate.Some of the properties of the enzymes involved were investigated in P. berghei preparations, utilizing a radioactive assay which measures the conversion of [7‐14C]pAB to [14C]H2pteroate. Apparent Km values of 0.28 μM for [7‐14C]pAB, 0.037 mM for pABG and 0.8 μM for hydroxymethyldihydropteridine were obtained. The reaction had absolute requirements for ATP and Mg++, and was stimulated by dithiothreitol. The enzymes required for the reaction were eluted together from Sephadex G‐200 columns in a molecular weight range of 200,000–250,000. In bacteria hydroxymethyldihydropteridine is converted 1st by a pyrophosphokinase to pyrophosphorylmethyldihydropteridine, and this compound is then condensed with pAB to form H2pteroate by H2pteroate synthetase. Both enzymic activities were demonstrated in P. berghei preparations and separated by DEAE‐Sephadex chromatography.The enzymic synthesis of H2pteroate by P. berghei is inhibited by several sulfonamides and diaminodiphenylsulfone (DDS). The latter compound is shown to be competitive with pAB, with a Ki value of 0.38 μM; pABG is also a competitive inhibitor. These data establish an enzymic basis of support for the evidence obtained in vivo which indicate that malaria synthesize their folate cofactors de novo. It is suggested that the antimalarial action of sulfonamides and DDS is due to their inhibition of plasmodial H2pteroate synthetase.