Mechanistic studies on the hydrolytic dehalogenation catalyzed by haloalkane dehalogenases are of importance for environmental and industrial applications. Here, Car-Parrinello (CP) and ONIOM hybrid quantum-mechanical/molecular mechanics (QM/MM) are used investigate the second reaction step of the catalytic cycle, which comprises a general base-catalyzed hydrolysis of an ester intermediate (EI) to alcohol and free enzyme. We focus on the enzyme LinB from Sphingomonas paucimobilis UT26, for which the X-ray structure at atomic resolution is available. In agreement with previous proposals, our calculations suggest that 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 EI. In the formation of the products, the protonated aspartic acid (Asp108) can easily adopt conformation of the relaxed state found in the free enzyme. 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 with respect to the water reaction is caused by strong stabilization of the reaction intermediate and transition state and their preorganization by electrostatic field of the enzyme.
(c) 2007 Wiley-Liss, Inc.