Combined QM/MM molecular dynamics simulations have been carried out for the dehalogenation reaction of the nucleophilic displacement of dichloroethane catalyzed by haloalkane dehalogenase. The computed chlorine kinetic isotope effects and free energies of activation in the wild-type and the Phe172Trp mutant enzyme are found to be consistent with experiment. In comparison with the uncatalyzed model reaction in water, the enzyme lowers the activation barrier by about 16 kcal/mol. The enormous enzymatic action was attributed to a combination of contributions from a change in the solvation effect and transition state stabilization. The unique features of tryptophan's ability to interact favorably with hydrophobic substrates and to form hydrogen bonds to the leaving group chloride ion at the transition state enable both factors to make significant contributions to the barrier lowering mechanism in the enzyme. This is in contrast to the reference reaction in water, in which hydrogen bonding interactions are weakened at the transition state because of dispersed charge distribution at the transition state relative to that in the reactant and product states.

Reactive flux molecular dynamics simulations have been carried out using a combined QM/MM potential to study the dynamics of the nucleophilic substitution reaction of dichloroethane by a carboxylate group in haloalkane dehalogenase and in water. We found that protein dynamics accelerates the reaction rate by a factor of 2 over the uncatalyzed reaction. Compared to the thermodynamic effect in barrier reduction, protein dynamic contribution is relatively small. However, analyses of the friction kernel reveal that the origins of the reaction dynamics in water and in the enzyme are different. In aqueous solution, there is significant electrostatic solvation effect, which is reflected by the slow reorganization relaxation of the solvent. On the other hand, there is no strong electrostatic coupling in the enzyme and the major effect on reaction coordinate motion is intramolecular energy relaxation.

Molecular dynamics simulations using a combined QM/MM potential have been performed to study the catalytic mechanism of human cathepsin K, a member of the papain family of cysteine proteases. We have determined the two-dimensional free energy surfaces of both acylation and deacylation steps to characterize the reaction mechanism. These free energy profiles show that the acylation step is rate limiting with a barrier height of 19.8 kcal/mol in human cathepsin K and of 29.3 kcal/mol in aqueous solution. The free energy of activation for deacylation step is 16.7 kcal/mol in cathepsin K and 17.8 kcal/mol in aqueous solution. The reduction of free energy barrier is achieved by stabilization of the oxyanion in the transition state. Interestingly, although the "oxyanion hole" has been formed in the Michaelis complex, the amide units do not donate hydrogen bonds directly to the carbonyl oxygen of the substrate, but they stabilize the thiolate anion nucleophile. Hydrogen bonding interactions are induced as the substrate amide group approaches the nucleophile, moving more than 2 angstroms and placing the oxy-anion in contact with Gln19 and the backbone amide of Cys25. The hydrolysis of peptide substrate shares a common mechanism both for the catalyzed reaction in human cathepsin K and the uncatalyzed reaction in water. Overall, the nucleophilic attack by Cys25 thiolate and the proton transfer reaction from His162 to the amide nitrogen are highly coupled, whereas a tetrahedral intermediate is formed along the nucleophilic reaction pathway.

No abstract