Purification and characterization of Plasmodium falciparum succinate dehydrogenase.
Succinate dehydrogenase (SDH), a Krebs cycle enzyme and complex II of the mitochondrial electron transport system was purified to near homogeneity from the human malarial parasite Plasmodium falciparum cultivated in vitro by FPLC on Mono Q, Mono S and Superose 6 gel filtration columns. The malarial SDH activity was found to be extremely labile. Based on Superose 6 FPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing-PAGE analyses, it was demonstrated that the malarial enzyme had an apparent native molecular mass of 90 +/- 8 kDa and contained two major subunits with molecular masses of 55 +/- 6 and 35 +/- 4 kDa (n = 8). The enzymatic reaction required both succinate and coenzyme Q (CoQ) for its maximal catalysis with Km values of 3 and 0.2 microM, and k(cat) values of 0.11 and 0.06 min(-1), respectively. Catalytic efficiency of the malarial SDH for both substrates were found to be relatively low (approximately 600-5000 M(-1) s(-1)). Fumarate, malonate and oxaloacetate were found to inhibit the malarial enzyme with Ki values of 81, 13 and 12 microM, respectively. The malarial enzyme activity was also inhibited by substrate analog of CoQ, 5-hydroxy-2-methyl-1,4-naphthoquinone, with a 50% inhibitory concentration of 5 microM. The quinone had antimalarial activity against the in vitro growth of P. falciparum with a 50% inhibitory concentration of 0.27 microM and was found to completely inhibit oxygen uptake of the parasite at a concentration of 0.88 microM. A known inhibitor of mammalian mitochondrial SDH, 2-thenoyltrifluoroacetone. had no inhibitory effect on both the malarial SDH activity and the oxygen uptake of the parasite at a concentration of 50 microM. Many properties observed in the malarial SDH were found to be different from the host mammalian enzyme.
Suraveratum
N
N
Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Krungkrai
S R
SR
Leangaramgul
P
P
Prapunwattana
P
P
Krungkrai
J
J
D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D051379:Mice; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009285:Naphthoquinones / Q000494:pharmacology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D013385:Succinate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D019802:Succinic Acid / Q000378:metabolism; D013804:Thenoyltrifluoroacetone / Q000494:pharmacology; D014451:Ubiquinone / Q000378:metabolism
D000962:Antimalarials; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D009285:Naphthoquinones; D014451:Ubiquinone; D013804:Thenoyltrifluoroacetone; D019802:Succinic Acid; D013385:Succinate Dehydrogenase; D010100:Oxygen; C014758:plumbagin