Biochemistry sEH has been measured experimentally.47 It is therefore possible that His523 may be either neutral (unprotonated) or ionized (protonated) in the active site of murine sEH. The protonation state of His523 has mechanistic implications because this residue is thought to be involved in the activation (deprotonation) of a water molecule prior to the hydrolysis step, which would require the Nε atom to be deprotonated. It has been suggested that His523 is deprotonated after the ring- opening reaction via a “proton shuttle”, in which the Nε proton is transferred to the deprotonated oxygen atom of Tyr465,37 but this mechanism has not yet been confirmed. As mentioned above, it has also been suggested that the hydroxyl groups of Tyr381 and Tyr465 do not lose their protons during the catalytic cycle,32 in which case the Nε proton could be transferred instead to the oxyanion formed upon ring opening. In the crystal structures that have been obtained for various forms of EH,31,48,49 the proximity of the Nε atom of His523 to the Asp333 side chain suggests that the Nε atom is protonated prior to the ring-opening reaction and donates a hydrogen bond to one of the Asp333 side chain oxygen atoms. In a MD study by Schiott et al.,37 it was found that NACs corresponding to nucleophilic attack of t-MSO by murine sEH were observed more frequently in a model with His523 protonated than in a neutral His model. The authors observed that protonation of His523 prevents Asp333 from forming H-bonds to other H-bond donors that otherwise would prevent nucleophilic attack of the epoxide. Hopmann et al. modeled the ring opening of t-MSO in a small model of sEH using density functional theory (DFT), with the His523 Nε atom being treated as neutral.50 This static model does not allow for the formation of hydrogen bonds between Asp333 and surrounding amino acid residues, as found in the MD study by Schiott et al.37 Hopmann et al. observed the transfer of a proton from the Tyr465 side chain to the epoxide oxygen, following the ring-opening step. A lower barrier (by 3.2 kcal/mol) was observed for nucleophilic attack at the epoxide carbon with the less-hindered methyl substituent, compared to that with a phenyl substituent. Hopmann et al. also modeled the hydrolysis of the ester intermediate and observed a barrier higher than that calculated for the ring-opening reaction, consistent with hydrolysis being the rate-limiting step in experimental studies.51 Despite the agreement between experimental observations and the calculations of Hopmann et al., their study does not conclusively identify the correct protonation state of His523. The effects of the protein environment are a key factor in the reactivity of sEH, and hence, a modeling method that takes these into account, such as QM/MM, is well-suited to this type of problem. In this study, the ring-opening reaction of two epoxide substrates in murine sEH is modeled. The substrates are trans-stilbene oxide (t-SO) and trans-diphenylpropene oxide (t-DPPO) (Figure 1). The regioselectivity of nucleophilic attack in t-DPPO has been determined experimentally, using 18O-labeled substrate, and found to occur preferentially (97%) at the phenyl carbon atom [i.e., C(1) in Figure 1].22 The two epoxide carbon atoms in t-SO are chemically equivalent, and hence, modeling the reaction with this substrate allows for determinants other than the relative reactivity of the two epoxide carbon atoms to be probed. The substrates chosen here allow us to test the ability of these QM/MM methods to predict regioselectivity and also investigate possible factors that control the regioselectivity of the ring-opening process. Here we model the nucleophilic attack by Asp333 at each of the two epoxide carbon atoms in t-SO and t-DPPO in multiple Article QM/MM (AM1/CHARMM22) umbrella sampling MD simulations. Extensive sampling, such as that used here, is very important when modeling selectivity in enzymes, as has been shown in recent studies.4,52 Relative barrier heights to nucleophilic attack at the two epoxide carbon atoms in t-DPPO are compared and found to be consistent with the experimentally observed regioselectivity.22 A large difference in the free energy barriers to nucleophilic attack for the two epoxide carbons atoms in t-SO is observed, despite the chemical equivalence of the two atoms. High-level ab initio QM calculations are used to correct the AM1 energies, yielding barriers that are in quantitative agreement with barriers derived from experimentally determined rate constants for similar reactions. The results are analyzed to identify the causes of this selectivity in the active site. Our QM/MM calculations reveal important interactions that influence the selectivity of ring opening, including the proximity of the epoxide carbon atoms to the nucleophile, the chemical environment of the epoxide carbon, and the hydrogen bonding interactions between the epoxide oxygen atom and the Tyr381 and Tyr465 side chains. These calculations provide insight into the factors that control stereoselectivity in epoxide hydrolase and may be of use in the design of novel catalysts.53 ■ COMPUTATIONAL METHODS Model Setup. The crystal structure of murine sEH without substrate (PDB entry 1CQZ)31 was obtained from the Protein Data Bank.54,55 In murine sEH, the epoxide hydrolase activity is conducted in the C-terminal catalytic domain (Val235− Ala544); hence, only this region is modeled. The coordinates of an inhibitor, N-cyclohexyl-N′-(3-phenyl)propylurea, from another sEH structure (PDB entry 1EK1)33 were docked into the active site of the catalytic domain of strand A. The inhibitor was modified to the required substrate, t-SO or t-DPPO (Figure 1). The S,S enantiomer of each substrate was modeled, as these have been shown experimentally in murine sEH to be turned over (slightly) faster than the corresponding R,R enantiomer.25 There are two possible orientations for t-DPPO in the active site, because of asymmetry about the epoxide ring, so simulations were performed for both of these orienta- tions (Figure 2). Hence, three enzyme−substrate complexes, Figure 2. Two different binding orientations of t-DPPO modeled in this study. The t-DPPO(1) and t-DPPO(2) orientations are colored blue and orange, respectively. The structures were obtained from unrestrained QM/MM MD simulations. termed t-SO, t-DPPO(1), and t-DPPO(2), are modeled. Similar approaches have been applied successfully in modeling 1776 dx.doi.org/10.1021/bi201722j | Biochemistry 2012, 51, 1774−1786