The isofunctional host enzyme should

The isofunctional host enzyme should be analyzed in parallel with the parasite enzyme according to structure based drug design approach in order to evaluate the selectivity and possible toxicity [15]. Therefore the gene encoding Bos taurus muscle enolase (BtEno3) which is counterpart of T. annulata enolase, was cloned, expressed, purified and biochemically characterized for the first time in the literature. As no empirically defined structure was solved, BtEno3 was modelled to predict 3-dimensional (3D) protein structure of the enzyme. The substrate 2‑phosphoglycerate (2PG) was docked into enzyme and molecular dynamics simulation of the enzyme-ligand complex was carried out to predict binding behavior of the substrate on the enzyme. These in vitro and in silico analyses on BtEno3 were performed to enlighten the further drug design studies to develop alternative drugs to be used in the treatment of tropical theileriosis.
Material and methods
Results and discussion
Conclusion The gene encoding the Bos taurus muscle enolase enzyme was successfully isolated and cloned in this study. Optimization of the cloning, gene Miconazole and purification was performed and protein elution at 95% purity was achieved. An alternative method has been proposed to eliminate impurities in the purification process, which plays an important role in the biochemical characterization of the enzyme. It has been achieved to eliminate the extra band formation which was a constant problem throughout this particular experimental study. For the first time in the literature, kinetic parameters of Bos taurus enolase 3, which plays a role in conversion of 2PG to PEP in the glycolytic pathway, was characterized by this study. In order to apply in silico studies, it is necessary to define the 3D structure of the enzyme. For this purpose, a homology model was built and the accuracy of this model was tested by web based evaluation and analysis programs. Substrate 2PG was docked into active site of the enzyme using molecular docking method for the analysis of interaction of enzyme-substrate complex. Enzyme-substrate complex built by homology modelling and molecular docking methods has been simulated by the molecular dynamics methods to stabilize structure and analyzed for its closeness to empirical results. The position of substrate and the final conformation of the complex were compared with the experimentally determined same molecules and their accuracy was analyzed. Overall, the reliable 3-D structure of the BtEno3-2PG complex is defined without the need of X-ray crystallography a time-consuming and expensive method and enzyme-substrate interactions are revealed. These in vitro and in silico analyses of the enolase from Bos taurus would enlighten further new drug development studies to be used in the treatment of theileriosis.
Acknowledgements This research has been supported by Yildiz Technical University Scientific Research Projects Coordination Department. Project Number: 2015-07-04-KAP07. The numerical calculations reported in this paper were fully/partially performed at TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources).