Purification and characterization of dihydroorotate dehydrogenase from the rodent malaria parasite Plasmodium berghei.
Dihydroorotate dehydrogenase (DHODase) has been purified 400-fold from the rodent malaria parasite Plasmodium berghei to apparent homogeneity by Triton X-100 solubilization followed by anion-exchange, Cibacron Blue F3GA-agarose affinity, and gel filtration chromatography. The purified enzyme has a molecular mass of 52 +/- 2 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of 55 +/- 6 kDa by gel filtration chromatography, and it has a pI of 8.2. It is active in monomeric form, contains 2.022 mol of iron and 1.602 acid-labile sulfurs per mole of enzyme, and does not contain a flavin cofactor. The purified DHODase exhibits optimal activity at pH 8.0 in the presence of the ubiquinone coenzyme CoQ6, CoQ7, CoQ9, or CoQ10. The Km values for L-DHO and CoQ6 are 7.9 +/- 2.5 microM and 21.6 +/- 5.5 microM, respectively. The kcat values for both substrates are 11.44 min-1 and 11.70 min-1, respectively. The reaction product orotate and an orotate analogue, 5-fluoroorotate, are competitive inhibitors of the enzyme-catalyzed reaction with Ki values of 30.5 microM and 34.9 microM, respectively. The requirement of the long-chain ubiquinones for activity supports the hypothesis of the linkage of pyrimidine biosynthesis to the electron transport system and oxygen utilization in malaria by DHODase via ubiquinones [Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].
Krungkrai
J
J
Laboratory of Medical Biochemistry, Rockefeller University, New York, New York 10021.
Cerami
A
A
Henderson
G B
GB
D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003902:Detergents; D004081:Dihydroorotate Oxidase / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008970:Molecular Weight; D017830:Octoxynol; D010962:Plasmodium berghei / Q000201:enzymology*; D011092:Polyethylene Glycols; D013455:Sulfur / Q000032:analysis
D003902:Detergents; D011092:Polyethylene Glycols; D013455:Sulfur; D017830:Octoxynol; D007501:Iron; D004081:Dihydroorotate Oxidase