A molecular modeling study of the catalytic mechanism of haloalkane dehalogenase. 2. Quantum chemical study of complete reaction mechanism

Haloalkane dehalogenase is a bacterial enzyme, which catalyzes hydrolytic cleavage of the carbon-halogen bond of haloalkanes. Quantum mechanical calculations at the semiempirical level using the MOPAC/ DRIVER methodology were applied to study the enzymatic hydrolysis.of 1,2-dichloroethane to 2-chloroethanol. In our previous study, the first S(N)2 step of dehalogenation reaction was investigated (Damborsky, J.; Kuty, M.; Nemec, M.; Koca, J. A Molecular Modeling Study df the Catalytic Mechanism of Haloalkane Dehalogenase: 1. Quantum Chemical Study of the First Reaction Step. J. Chem. Inf: Comput. Sci. 1997, 37, 562-568). The present contribution explores the complete three-step reaction to determine the rate-limiting reaction step and to investigate the importance of active site residues for the kinetics and thermodynamics of the hydrolysis. The nucleophilic addition (Ad(N)) step has the highest energy barrier, which is in qualitative agreement with experimental rates, assigning the second hydrolytic step as the rate-limiting one. In order to establish the catalytically important active-site residues, Mulliken charges of selected active-site atoms were monitored along the reaction pathway. A significant change in charges on the hydrogen atoms of Trp(125), Trp(175), and Phe(172) active-site residues was observed. These residues interact with the halide ion released during the S(N)2 step. Changes in charges on the hydrogen atoms of Trp(125), and Glu(56) prove the significance of those residues in the stabilization of the partial charge developed on the oxygen atom of the nucleophilic aspartate (Asp(124)). The same methodology confirmed the importance of the charge relay system (Asp(124), His(289), and Asp(260) residues) in the base-hydrolysis reaction (Ad(N) step).