Molecular dynamics simulations of ground and transition states have been carried out to 540 ps for the S(N)2 displacement of Cl- from 1,2- dichloroethane (DCE) by the Asp124-CO2 - at the active site of the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The nucleophilic carboxylate of Asp 124-CO2 - is electrostatically stabilized in the enzyme-substrate (ES) complex by hydrogen bonding. OD1 of Asp124-CO2 - is hydrogen bonded to water396 and the backbone amide hydrogens of Glu56 and Trp125. The nucleophilic oxygen (OD2) of Asp124-CO2 - is hydrogen bonded to water323. This is a stable but nonreactive conformation. The kinetically essential near attack conformations (NACs) are formed when water323 dissociates to allow the C(1) of the gauche conformation of DCE to be within ~3 Å from the nucleophilic OD2 of Asp124-CO2- while Cl(1) is hydrogen bonded to the indole NH of Trp 125. By comparing the molecular dynamics simulations for the ES complex and the enzyme transition-state (TS) structure, one can observe the changes in the active site structure in the course of the reaction. In contrast to the NAC with a single hydrogen bond between Trp125 and Cl(1), the TS has two hydrogen bonds to the leaving Cl(1), the Trp125 hydrogen bond (2.43 ± 0.29 Å) and the Trp175 hydrogen bond (2.24 ± 0.19 Å). Assistance of the two tryptophan hydrogen bonds to lowering the activation energy may be about 2 kcal/mol. Certain hydrogen bonds are critical to maintaining the tertiary structure of the active site and essential functions of water molecules in the reaction. On comparing the NAC structures to the transition-state structures, hydrogen-bonding changes are seen. These include the already-mentioned electrostatic interaction of Trp 175 with Cl(1) in the TS. In the TS, we also observe the formation of a tight hydrogen-bonding matrix involving the water323, water392, and water396. This matrix exactly positions water392 such that it (i) is a member of the triad Asp260-CO2 -···H-(δ)N-His289-(ε)N···water392 and (ii) is aligned correctly to act as a nucleophile toward the carbonyl of the alkyl-ester intermediate (Asp124-CO2-CH2CH2-Cl), formed upon departure of Cl-. In other words, the ground state of the second enzymatic reaction is set up on reaching the transition state of the first reaction. The hydrogen bond between OD2 of Asp260-CO2- and H-(δ)N of His289 exists in both ES and TS structures. Aside from being a part of the catalytic triad for the second reaction, this hydrogen bond is responsible for maintaining the active site structure. Disruption of this hydrogen bond by moving the imidazole proton from H-(δ)N to create H-(ε)N of His289 brings about deep-seated changes in the ES ground state such that NACs are not formed. This hydrogen bond is similar to those between Asp and His in the serine esterases where it may also play a role in the stability of the active site.
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