The formation of enzyme-ligand complex that has an important role in enzymatic reactions was simulated by MD simulation for the interaction of Candida Antarctica Lipase B (CALB) with dimethyl methylphosphonate (DMMP) and methylcaprylate (MEC) ligands. We found that this complex was formed for these ligands and so the reaction between CALB and them can be occurred. Afterward by applying the quantum mechanics calculations with the B3PW91 computational method, we have described complete catalytic reaction mechanism of CALB inhibition and aging with DMMP and determined its multistep free-energy reaction profiles for the first time. It was shown that in phosphonylation process, which contains a two-step addition-elimination reaction, the addition step formed a trigonal pyramidal intermediate by passing from the free energy barrier of 17.2 kcal mol-1 while elimination of methanol molecule with 5.5 kcal mol-1 free energy made the inhibited enzyme. The second step of the reaction was examined for two possible pathways; dephosphonylation and aging of inhibited enzyme. From the inhibited enzyme construction to the second trigonal pyramidal intermediate, both pathways passed from a nucleophilic attack of a water molecule on the attached phosphoryl group, which needed 15.8 kcal mol-1 to pass from free energy barrier. Dissociation between the phosphoryl group and the oxygen atom of the Ser105 side chain led to dephosphonylation, which required 12.3 kcal mol-1 free energy to happen. Nevertheless, the breakdown of the covalent bond between phosphoryl group and methoxy group make possible enzyme aging and wanted 9.8 kcal mol-1 activation free energy. It was exposed that the aging reaction was energetically and kinetically preferred and under real biological conditions, the aging of the CALB active site by DMMP can take place. It was determined that the enzymatic reaction here was influenced strongly by the hydrogen bond interactions.

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