| Understanding the molecular origin of enzyme catalysis and providing an atomistic view of the mechanism of enzyme action are the major focus of this thesis. Molecular dynamics simulations using a combined quantum mechanical molecular mechanical method (QM/MM) has been utilized to estimate the free energy of the various steps involved in the catalytic mechanism of the enzyme, Haloalkane Dehalogenase (DhlA). DhlA studied in this thesis is from Xanthobacter Autotrophicus GJ10 and it hydrolyses carbon-halogen bonds of a broad range of n-haloalkanes. Hence this enzyme is of great importance from an environmental perspective. The halogenated hydrocarbons are converted to their corresponding alcohols via a three step process. The study presented herein concentrates on the estimation of the activation barriers of the three steps involved in converting dichloroethane (DCE), a natural substrate of the enzyme, into 2-chloroethanol and elucidating the role of crucial active site residues in enzyme catalysis. Additionally, an important aspect of enzymatic reactions namely, the pKa values of ionizable residues is studied using a novel combined QM/MM titration method. The new methodology is tested by reproducing the experimentally known pKa values of ionizable residues in solution. Further, the method is successfully extended to evaluating absolute pKa values of ionizable residues in tri-peptides. |
