The importance of reactant positioning in enzyme catalysis: a hybrid quantum mechanics/molecular mechanics study of a haloalkane dehalogenase

Abstract

Hybrid quantum mechanics/molecular mechanics calculations using Austin Model 1 system-specific parameters were performed to study the S(N)2 displacement reaction of chloride from 1,2-dichloroethane (DCE) by nucleophilic attack of the carboxylate of acetate in the gas phase and by Asp-124 in the active site of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The activation barrier for nucleophilic attack of acetate on DCE depends greatly on the reactants having a geometry resembling that in the enzyme or an optimized gas-phase structure. It was found in the gas-phase calculations that the activation barrier is 9 kcal/mol lower when dihedral constraints are used to restrict the carboxylate nucleophile geometry to that in the enzyme relative to the geometries for the reactants without dihedral constraints. The calculated quantum mechanics/molecular mechanics activation barriers for the enzymatic reaction are 16.2 and 19.4 kcal/mol when the geometry of the reactants is in a near attack conformer from molecular dynamics and in a conformer similar to the crystal structure (DCE is gauche), respectively. This haloalkane dehalogenase lowers the activation barrier for dehalogenation of DCE by 2-4 kcal/mol relative to the single point energies of the enzyme's quantum mechanics atoms in the gas phase. S(N)2 displacements of this sort in water are infinitely slower than in the gas phase. The modest lowering of the activation barrier by the enzyme relative to the reaction in the gas phase is consistent with mutation experiments.

Scheme 1

Figure 1

A shows the geometry of acetate and DCE as found in enzyme NAC and used in the ab initio calculations of the interaction energies. B shows the relative ab initio optimized geometries of the reactants obtained without angular contraints.

Figure 2

Starting conformations of DCE in the active site of DhlA. A has the DCE is the same position as found in the crystal structure of the enzyme⋅substrate complex (COMF2). Structure in B is the last coordinate set from a molecular dynamics simulation of DhlA starting with the same crystal structure (COMF1). The conformation of DCE in both structures is gauche. The dashed lines show the interaction between OD2 of Asp-124 and C2 of DCE (OD2⋅⋅⋅C2).

Figure 3

Gas-phase structures of DCE undergoing nucleophilic attack by acetate. Structures in A have no dihedral constraints. Structures in B have dihedral constraints to restrict the attacking geometry of acetate to one similar to the enzyme. Parenthetical values are in angstroms and correspond to the distance between the attacking carboxylate oxygen of acetate to the carbon of DCE to the carbon of DCE and the leaving chloride (C⋅⋅⋅O, C⋅⋅⋅Cl).

Scheme 2

Figure 4

Transition-state structure for COMF1 in the active site of DhlA. Dashed lines indicate hydrogen bonds formed between the enzyme and DCE and side chain of Asp-124. The hydrogen- bonding arrangement in COMF2 is similar to that depicted.

Figure 5

Picture of aromatic residues in contact with Trp-125 and Trp-175 in the active site of DhlA.