D-3-hydroxybutyrate oxidation in mitochondria by D -3-Hydroxybutyrate dehydrogenase in Tetrahymena pyriformis


Omar Akil , Zakaria El Kebbaj, Norbert Latruffe * and Mâ??Hammed Saïd El Kebbaj

Tetrahymena pyriformis a ciliated protozoan, is considered as a good indicator of water pollution. However its energy supply is poorly understood. This work was focused on the metabolism of hydroxybutyrate through the study of the membrane bound mitochondrial NAD+ -dependent D-3- hydroxybutyrate dehydrogenase (EC. (BDH), a ketone body catalysing enzyme involved in the interconversion of D-3- hydroxybutyrate to acetoacetate. Due to lack of informations, the physicochemical properties and kinetic parameters of the enzyme were examined. The results are the following: 1) D-3-hydroxybutyrate is a good substrate for mitochondria. 2) The enzyme catalytic process follows a bi bi-ordered mechanism where the coenzyme binds first, then allowing the substrate linkage to the active site. 3) Two optimal pH values of 8 and 6.5 corresponding to D-3-hydroxybutyrate oxidation and to acetoacetate reduction respectively. On the other hand, pH changes affect the coenzyme binding to the active site. 4) The BDH activity was found strongly linked to submitochondrial vesicles indicating that the protozoan enzyme is membranous and could require lipids for its function as well as it is for the mammalian enzyme. Moreover, an optimal temperature (40°C) and a break appearing in the Arrhenius plot at 19°C were found. The break suggests a membrane lipid fluidity-dependency of BDH conformational change. 5) Several ligands of the active site including methylmalonate and succinate modulate the BDH activity and are competitive inhibitors toward D-3-hydroxybutyrate. 6) Divalent cations, Mg2+, Mn2+ and Zn2+ protect BDH against thermal inactivation. The protection is the strongest in the presence of Zn2+. Moreover, Ca2+ and Mg2+ are enzyme activators and modulate the substrate binding to the active site. On the other hand, EDTA, a chelating agent, inhibits the enzyme but prevents inhibition by substrate excess. This work provides new insights on the energy metabolism of T. pyriformis wild strain where D-3-hydroxybutyrate is a choice substrate where the properties of BDH have been established especially the activating role of non heavy divalent cations.

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