Computational insight into the glycosidase enzyme using bioinformatics tools and molecular dynamic simulations.

M. Pharm. University of KwaZulu-Natal, Durban 2013.One of the subjects focused on by theoretical and computational studies is that of the molecular systems. Molecular dynamic simulations can provide useful insights into the dynamic, thermodynamic and structural properties of the system. It is also a convenient way to study enzyme reactions. The glycoside hydrolase enzyme xylanase, a family GH-11 enzyme, hydrolyses xylan with a net retention of anomeric configuration via a double displacement mechanism. Catalytic residues Glu78 acts as a nucleophille and Glu172 as the general acid/base. In its catalytic cycle, the enzyme passes through different phases, three in particular: the free enzyme phase, the covalent enzyme-substrate complex and the non-covalent substrate in the enzyme pocket. All-atom molecular mechanic MD simulations were performed on the enzyme at each of the above phases for a total of 10ns, to study the effect of the substrate binding on the configuration and movement of the thumb-finger region. Various metrics were implemented to estimate the dynamics of this movement. The study has shown that the covalently bound substrate shows a shortening of the thumb-finger distance, assumedly to hold the substrate in the cleft for the reaction to occur, compared to the non-covalent system in which the distance widens at the beginning of the pathway to allow the substrate into the active site cleft and at the end of the process to expel the substrate. The enzyme was also investigated using binding free energy calculations, MMGBSA and MMPBSA, on 2ns simulation at all-atom MM level as well as AM1 level of description of the QM/MM simulation. Calculations were performed on the wild-type, Glu78Asp, Glu172Asp, Tyr69Phe, Tyr80Phe and Arg112Asn mutants. Results show that the catalytic residues Glu78 and Glu172 do not play a role in substrate binding yet they are crucial for the catalytic mechanism. The Tyr69 residue however, plays an important role in binding, since it is the hydrogen bond that stabilizes the boat conformation that is essential to reach TS. Tyr80 and Arg112 similarly play a role in binding. Future calculations that are in progress are QM/MM simulations at PM3, DFTB and RM1 level of description to verify the accuracy of each method. The information from this study sheds further light on factors that affect the catalytic process of the glycoside hydrolase enzyme such as the thumb-finger dynamics as well as the role played by some key residues in substrate binding, and can be used to aid the design of potential inhibitors