Dihydrofolate Reductase of Streptococcus f aecium II. PURIFICATION AND SOME PROPERTIES OF TWO DIHYDROFOLATE REDUCTASES FROM THE AMETHOPTERIN-RESISTANT

From a single amethopterin-resistant organism, Streptococcus faecium var. durans strain A, two different dihydrofolate reductases have been obtained as essentially homogeneous proteins in good yield. One of the reductases has a similar substrate specificity and turnover number (about 8000 moles per min per mole of enzyme) to the single reductase found in the amethopterin-sensitive strain of S. faecium var. durans, ATCC 8043, and has therefore been designated “wild type.” The other enzyme, which is distinguished by its ability to catalyze the reduction of folate, in addition to dihydrofolate, and by its lower turnover number (about 900 with dihydrofolate), has been designated “mutant type.” Since the wild type and mutant type reductases have sedimentation constants (sZO,buffer) of 2.58 S and 2.04 S, respectively, they are probably significantly different in molecular weight. Each exhibits a single pH optimum at pH 5.8 and is inactivated by urea. Neither is affected by methyhnercuric salts but the wild type reductase is inactivated by phenylmercuric acetate and P-mercuribenzoate. Monovalent cations increase the activity of the mutant type reductase but decrease that of the wild type reductase. It is suggested that the amethopterin resistance in vivo of strain A depends at least partly on the folate reductase activity of the mutant type reductase.