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

Hybrid quantum mechanics/molecular mechanics calculations using Austin Model 1 system-specific parameters were performed to study the SN2 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. SN2 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.

Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase

The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 1010-fold: but how? Our structures of the yeast PPase product complex at 1.15 Å and fluoride-inhibited complex at 1.9 Å visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (Onu) directly attacks PPi with a pKa vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and Onu, in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography.