Kinetic characterization of dihydrofolate reductase from Drosophila melanogaster.
The kinetic characteristics of a purified insect dihydrofolate reductase (DHFR) have been described. The Km values for the substrate dihydrofolate and the cofactor NADPH have been estimated by primary and secondary Hanes plots to be 0.3 and 5.2 microM, respectively. Drosophila melanogaster DHFR can use folate and NADH at acidic pH values, but at a much lower rate than the preferred substrate and cofactor. Folic acid is a partial competitive inhibitor of Drosophila DHFR (Ki = 0.4 microM) and trimethoprim is a complete competitive inhibitor (Ki = 5.4 microM). Methotrexate binds less tightly to the Drosophila enzyme than to many other DHFRs (Kd = 0.9 nM). Drosophila DHFR is inhibited by KCl and organic mercurials and is slightly activated by urea. These data indicate that Drosophila DHFR has some characteristics which are typical of vertebrate DHFRs and others which are typical of prokaryotic DHFRs. The study of this enzyme, therefore, should aid in the definition of the structural features that are responsible for the kinetic characteristics in different DHFRs.
Rancourt
S L
SL
Department of Biology, Queen's University, Kingston, Ont., Canada.
Walker
V K
VK
D000818:Animals; D004331:Drosophila melanogaster / Q000201:enzymology*; D005492:Folic Acid / Q000378:metabolism; D005493:Folic Acid Antagonists; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009941:Organomercury Compounds / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D005493:Folic Acid Antagonists; D009941:Organomercury Compounds; D009249:NADP; D011189:Potassium Chloride; D014508:Urea; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase