Purification and characterization of enzyme succinate dehydrogenase in Plasmodium falciparum T9 isolate

Abstract

Succinate dehydrogenase (SDH) functions in all aerobic organisms, as a membrane-bound enzyme and a component of both tricarboxylic acid cycle and complex II in electron transport chain. SDH catalyzes the oxidation of succinate to fumarate and is able to transfer the reducing equivalents directly to coenzyme Q (CoQ). The enzyme SDH is composed of four polypeptides. The large subunit which has molecular weight (Mr) of about 70 kilodltons (KDa) contains covalently bound FAD, namely flavoprotein (Fp). The small subunit has Mr of about 30 KDA, namely iron sulfur protein (Ip). The other two small hydrophobic polypeptides, cytochrome b subunits with Mr of about 15 and 13 KDa, anchor the Fp and Ip subunits to mitochondrial inner membrane and provide the binding site for CoQ. This study is focused on the aspect of purification and characterization of the enzyme SDH in P. falciparum (T9 isolate) and expected that the results will provide a basis for the development of novel antimalarial drug. Malarial SDH was purified by using three-step purification protocol starting from the supernatant fraction of P. falciparum (T9 isolate). These included Mono Q anion-exhange, Mono S cation-exchang and Superose 6 gel filtration chromatography on a fast protein liquid chromatographic system. The malarial SDH was locate in cytosol, and found to be extremely labile. It was more stable at -196℃ than -20℃. The purified enzyme had native Mr of 86-91 KDa. By SDS-PAGE, the malarial SDH revealed two subunits with Mr of 56.4 ± 3.4 KDa for Fp subunit and 35.0 ± 1.7 KDa for Ip subunit. The apparent Michaelis-Menten constants (Km) and turnover number (kcat) for succinate were 3.01 µM and 0.11 min-1, respectively ; for CoQo they were 0.20 µM and 0.06 min-1. Fumarate, the product of 80.99 µM. Malonate and oxaloacetate were substrate analog inhibitors with their Ki values of 13.02 and 12.06 µM, respectively. Plumbagin was found to inhibit more than 50 at a concentration of 5 µM. Antimalarial drugs, such as chloroquine, artemisinine and atovaquone have no effect on the malarial SDH. It was relatively insensitive to 2-thenoyltrifluoroacetone. Many properties observed in the malarial SDH were different from the host mammalian enzyme.