Jaroslaw Pawlak1, Brian J. Bahnson2, Vernon E. Anderson1
1Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935, USA,
2Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA
The enoyl-CoA hydratase catalyzed hydration of a,b-unsaturated thiolesters has been modeled by using the crystal structure of 4-(N,N,-dimethylamino)cinnamoyl-CoA bound at the active site. The quantum chemical calculation used the ONIOM mixed level procedure to permit the substrate thiolester and water molecule to be modeled using B3LYP/6-31G(d) level of theory and the active site residues modeled at a semiempirical level using the PM3 Hamiltonian. The results permitted the identification of a stable thiolester enolate intermediate, supporting a stepwise reaction mechanism. The calculation also suggests that the same proton removed from the nucleophilic water molecule is transferred to Ca in the subsequent protonation of the enolate intermediate. This observation reconciles the stepwise mechanism with the previously reported double isotope effect study.