Biological significance of phosphoenolpyruvate carboxykinase in a cestode parasite, Raillietina echinobothrida and effect of phytoestrogens on the enzyme from the parasite and its host, Gallus domesticus.

Phosphoenolpyruvate carboxykinase (PEPCK) is involved in glycolysis in the cestode parasite, Raillietina echinobothrida; whereas, it executes a gluconeogenic role in its host, Gallus domesticus. Because of its differing primary function in the cestode parasite and its host, this enzyme is regarded as a plausible anthelmintic target. Hence, the biological significance of PEPCK in the parasite was analysed using siRNA against PEPCK from R. echinobothrida (RePEPCK). In order to find out the functional differences between RePEPCK and GdPEPCK (PEPCK from its host, G. domesticus), PEPCK genes from both sources were cloned, over-expressed, characterized, and some properties of the purified enzymes were compared. RePEPCK and GdPEPCK showed a standard Michaelis-Menten kinetics with K mapp of 46.9 and 22.9 µ m, respectively, for phosphoenolpyruvate and K mapp of 15.4 µ m for oxaloacetate in GdPEPCK decarboxylation reaction. Here, we report antagonist behaviours of recombinant PEPCKs derived from the parasite and its host. In search of possible modulators for PEPCK, few phytoestrogens were examined on the purified enzymes and their inhibitory constants were determined and discussed. This study stresses the potential of these findings to validate PEPCK as the anthelmintic drug target for parasitism management.

Biocomputational analysis of phosphoenolpyruvate carboxykinase from Raillietina echinobothrida, a cestode parasite, and its interaction with possible modulators.

Phosphoenolpyruvate carboxykinase (PEPCK) involved in gluconeogenesis in higher vertebrates opposedly plays a significant role in glucose oxidation of the cestode parasite, Raillietina echinobothrida. Considering the importance of the enzyme in the parasite and lack of its structural details, there exists an urgent need for understanding the molecular details and development of possible modulators. Hence, in this study, PEPCK gene was obtained using rapid amplification of cDNA ends, and various biocomputational analyses were performed. Homology model of the enzyme was generated, and docking simulations were executed with its substrate, co-factor, and modulators. Computer hits were generated after structure- and ligand-based screening using Discovery Studio 4.1 software; the predicted interactions were compared with those of the existing structural information of PEPCK. In order to evaluate the docking simulation results of the modulators, PEPCK gene was cloned and the overexpressed protein was purified for kinetic studies. Enzyme kinetics and in vitro studies revealed that out of the modulators tested, tetrahydropalmatine (THP) inhibited the enzyme with lowest inhibition constant value of 93 nm. Taking the results together, we conclude that THP could be a potential inhibitor for PEPCK in the parasite.

Differential kinetics at PK/PEPCK branch point in the cestode, Raillietina echinobothrida.

Pyruvate kinase (PK; EC 2.7.1.40) and phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) are essential regulatory enzymes of glucose oxidation in helminths, the PK/PEPCK branch point being the first divergent step between carbohydrate catabolism of the parasites and their hosts. Recently, PEPCK from the cestode parasite, Raillietina echinobothrida, has been purified and characterized. In order to find out the differential kinetics, if any, at PK/PEPCK branch point in the parasite, in this study, we purified and characterized the parasite PK and compared it with the parasite PEPCK. The purified PK displayed standard Michaelis-Menten kinetics with Kmapp of 77.8 µM for its substrate PEP, whereas the Kmapp was 46.9 µM for PEPCK. PEP exhibited differential kinetics at PK/PEPCK branch point of the parasite and behaved as a homotropic effector for PEPCK, but not for PK. The inhibitory constant (Ki) for genistein and daidzein (phytochemicals from Flemingia vestita) was determined and discussed. From these results, we hypothesize that PK/PEPCK branch point is a probable site for anthelmintic action.