During the first step of the reaction, the primary alcohol from Ser105, which is the nucleophilic centre of the catalytic triad, attacks the ester bond of DMFDCA, forming the first tetrahedral intermediate (TI) stabilized by the three hydrogen bonds located in the oxyanion hole. During this process, the imidazole group of His224 acts as a general base and accepts a proton from Ser105, thereby activating it and enhancing its nucleophilicity, while Asp187 stabilizes the His224 residue by forming a hydrogen bond with it. In the second step, methanol is released, and the acyl-enzyme complex (AEC) is formed. Methanol is continuously removed via evaporation, making the reaction irreversible. In the third step, the hydroxyl group from α,ω-ALD, and DLD interacts with AEC, creating the second tetrahedral intermediate, again stabilized by the oxyanion hole. In the final step, the enzyme is deacylated, and the ester product is formed.