The Occurrence and Properties of Pteridine Reductase: Dihydrofolate Reductase in Crithidia fasciculata

Abstract

Pteridine reductase: dihydrofolate reductase obtained from Crithidia fasciculata was purified 60-fold. The molecular weight was estimated to be 110,000 daltons by Sephadex G-150 gel filtration. The enzyme reduced the neopterin isomers (l-threo-, l-erythro-, D-threo- and d-erythro-), 6-hydroxymethylpterin, 6-methylpterin and xanthopterin as well as dihydrofolate and dihydro-pteroate. The reaction with l-threo-neopterin had a double pH optimum (6.0 and 4.5), while that with 6-hydroxymethylpterin occurred over a pH range between 6.5 and 4.5. The optimum pH’s using dihydrofolate and dihydropteroate as the substrates were 6.8 and 7.0, respectively. Km values for l-threo-neopterin, 6-hydroxymethylpterin, dihydrofolate and dihydropteroate were 3.5, 3.4, 4.8 and 0.9 μM, respectively. The reaction was dependent on NADPH, requiring two mol of NADPH for reduction of one mol of l-threo-neopterin. Km values for the NADPH in assays with l-threo-neopterin, 6-hydroxymethylpterin, dihydrofolate and dihydropteroate were 11, 5.9, 5.9 and 2.1 μM, respectively. The reaction product was the tetrahydro form of each pteridine compound. Enzyme activity was inhibited by biopterin, folate, methotrexate, pyrimethamine, trimethoprim and NADP, as well as by p-chloromercuribenzoate, N-ethylmaleimide and urea. These evidences suggest that this enzyme is a new type of dihydrofolate reductase. Thus, the name, pteridine reductase: dihydrofolate reductase, is suggested for this enzyme.