Kinetic and molecular properties of dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi.
Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) was partially purified from a cloned strain of pyrimethamine-sensitive Plasmodium chabaudi and a drug-resistant clone derived from it. A molecular weight of approximately 120000 was estimated by gel filtration for enzyme from both pyrimethamine-sensitive and resistant parasites. The specific activities of the crude enzyme at pH 7.4 were 2.7 +/- 0.8 and 1.4 +/- 0.6 nmol min-1 mg-1 protein for sensitive and resistant strains, respectively. Methotrexate titration (pH 7.4, 37 degrees C) indicated that the apparent turnover number of the enzyme from the sensitive parasites was 1229 +/- 322 mol min-1 mol-1 compared with 1238 +/- 179 mol min-1 mol-1 for the enzyme from the resistant parasites. There was therefore no significant difference in the amounts of the enzyme from both sources. The Km value for dihydrofolate (9.3 microM) of the enzyme from the drug-sensitive parasites at pH 7.4 was lower than that from the resistant parasites by a factor of approximately 4. The Km values for NADPH of the enzyme from both sources were similar. Inhibition by pyrimethamine of the enzyme from the sensitive parasites was competitive with dihydrofolate, with Ki of 0.26 nM. By contrast, noncompetitive inhibition was observed for the enzyme from the resistant parasites, with Kis of 50 nM and Kii of 33 nM. The enzyme from drug-sensitive and drug-resistant parasites had different activity profiles with respect to pH and temperature. Moreover, the former was more sensitive to heat denaturation than the latter. From these results, it was concluded that the major basis for drug resistance is not an increase in enzyme content, but a large decrease in drug binding with the structurally different enzyme.
D000818:Animals; D004351:Drug Resistance; D005492:Folic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010961:Plasmodium / Q000187:drug effects / Q000201:enzymology*; D011739:Pyrimethamine / Q000494:pharmacology*; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
C010920:dihydrofolate; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D011739:Pyrimethamine