Second Step of Hydrolytic Dehalogenation in Haloalkane Dehalogenase Investigated by QM/MM Methods
Otyepka, M., Banas, P., Magistrato, A., Carloni, P., Damborsky, J.
PROTEINS 70: 707-717 (2008)
We investigate mechanism and energetics of the hydrolytic dehalogenation catalyzed by haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 by Car-Parrinello (CP) and ONIOM hybrid quantum-mechanical/molecular mechanics (QM/MM) simulations, QM calculations and classical molecular dynamics. We focus on the second reaction step of the catalytic cycle, which comprises a general base-catalyzed hydrolysis of an ester intermediate to alcohol and free enzyme. In this step, a histidine residue (His272), polarized by glutamate (Glu132), acts as a base, accepting a proton from the catalytic water molecule and transferring it to an alcoholate ion. The reaction proceeds through a metastable tetrahedral intermediate, which shows an easily reversed reaction to the ester intermediate. The overall free energy barrier of the reaction calculated by potential of the mean force integration using CP-QM/MM calculations is equal to 19.5±2 kcal.mol-1. The lowering of the energy barrier of catalyzed reaction is caused by strong stabilization of the reaction intermediate and transition state by local electrostatic field of the enzyme, while its intrinsic dynamics plays a minor role. In the formation of the products, the protonated aspartic acid (Asp108) can easily adopt conformation of the relaxed state found in the free enzyme.