Second Step of Hydrolytic Dehalogenation in Haloalkane Dehalogenase Investigated by QM/MM Methods

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Authors

OTYEPKA Michal BANÁŠ Pavel MAGISTRATO A. CARLONI P. DAMBORSKÝ Jiří

Year of publication 2008
Type Article in Periodical
Magazine / Source Proteins: Structure, Function, and Bioinformatics
MU Faculty or unit

Faculty of Science

Citation
Web http://loschmidt.chemi.muni.cz/peg/abstracts/prot07b.html
Field Biochemistry
Keywords dehalogenase; LinB; Sphingomonas paucimobilis UT26; quantum-mechanical/molecular mechanics (QM/MM) simulations
Description 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.
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