Enoyl-CoA hydratase (ECH) catalyzes the second step in the vital β-oxidation pathway of fatty acid metabolism. This enzyme catalyzes the syn-addition of a water molecule across the double bond of 4-(N,N-dimethylamino) cinnamoyl-CoA (DAC-CoA). In this work, the reaction mechanisms of ECH were investigated using the density functional theory (DFT) methods. The different protonation states in which the important residues Glu164 and Glu144 are either neutral or ionized were considered. Four models of the active site were designed based on the X-ray crystal structure of the enzyme. The calculations gave strong support to the proposed mechanism and confirmed that both Glu164 and Glu144 are in a deprotonated state in the reaction mechanism of ECH. In addition, we constructed a model of the active site with the inhibitor acetoacetyl-CoA based on the crystal structure. Caomparison of the calculated energy barriers showed that binding of the keto-enol form of the inhibitor is more reasonable than that of the di-keto form in the inhibition process. Moreover, acetoacetyl-CoA was found to exhibit a keto-enol tautomerism when it acts as an inhibitor in the reaction. The present theoretical results indicated that both residues Glu164 and Glu144 are unprotonated in ECH with the substrate bound, while only Glu164 is unprotonated when the inhibitor binds ECH.

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