Biochemistry preference for attack at C(1) in the protonated models and at C(2) in the neutral models, albeit to a lesser extent for the latter. The higher barriers for attack at C(1) in the neutral His523 models could be due to the lack of a hydrogen bonding interaction between the Tyr465 side chain and epoxide oxygen atom, which has been suggested to be important in stabilizing the TS.77 In the unprotonated models, the Tyr465 side chain donates a hydrogen bond to the Asp333 Oδ2 carboxylate atom in the substrate complex. In contrast, the Tyr465 side chain donates a hydrogen bond with the epoxide oxygen atom in the protonated models (Figure 4). As the nucleophilic attack Figure 4. Active site structures from AM1/CHARMM22 umbrella sampling MD simulations of sEH complexed with t-SO for two systems differing in the protonation state of His523: one in which His523 is protonated at the Nε atom and positively charged (green) and one in which His523 is not protonated at the Nε atom and is neutral (purple). The structures correspond to the reactant complexes, in which r = −2.0 Å. In the former, a hydrogen bond is observed between the His523 Nε proton and Oδ2 atom of Asp333. In the latter, a hydrogen bond is observed between the Oδ2 atom of Asp333 and the phenol hydrogen of Tyr465. Important hydrogen bonds are shown by dotted lines. progresses [at either C(1) or C(2)] and the TS is formed, this hydrogen bond to the Asp333 Oδ2 atom is lost, but the Tyr465 hydroxyl group remains unable to donate a hydrogen bond to the epoxide oxygen atom. The presence of a hydrogen bond from Tyr465 to the epoxide oxygen not only stabilizes the covalent ring-opened intermediate but also lowers the barrier to its formation, because of stabilization of the transition state, as is discussed below. The protonated Nδ atom of His523 donates a hydrogen bond to the Oδ2 atom of Asp495, another member of the catalytic triad, for all simulations of all models (according to the hydrogen bond definitions detailed below). Asp495 has been postulated to be involved in a “charge-relay” mechanism during the hydrolysis step.50 Given the proximity of the His523 Nε and Asp333 Oδ2 atoms in the crystal structure (2.41 Å31) and the fact that the experimentally observed regioselectivity for the ring-opening reaction in t-DPPO is only correctly predicted in the simulations in which the Nε atom is protonated, it seems most likely that the His523 side chain is protonated at both the Nδ and Nε positions during the initial stages of the sEH-catalyzed reaction. For this reason, only the simulations in which the His523 Nε atom is protonated (i.e., positively charged) are discussed further. Free Energy Barriers. To increase the amount of conformational sampling for the free energy profiles, additional AM1/CHARMM22 umbrella sampling calculations were performed on the t-SO, t-DPPO(1), and t-DPPO(2) model Article systems. The barriers obtained from these simulations (given in Table 3) indicate the same predicted regioselectivity as the free Table 3. QM/MM Free Energy Barriers (in kilocalories per mole) for the Nucleophilic Attack at Each of the Epoxide Carbons, C(1) and C(2), from the Extended Umbrella Sampling MD Simulations of the t-SO, t-DPPO(1), and t- DPPO(2) Model Systems ΔG⧧(P) t-DPPO(1) t-SO t-DPPO(2) QM method C(1) C(2) C(1) C(2) C(1) C(2) AM1a 22.7 30.1 19.0 25.0 23.4 30.7 B3LYP/6-31G(d) 7.3 16.6 9.7 11.0 6.8 13.9 SCS-MP2/(aug)-cc- 12.3 19.9 12.8 15.8 13.2 21.8 pVTZ aEnergy barriers calculated from extended umbrella sampling simulations with His523 protonated at the Nε position. energy barriers from the shorter simulations shown in Table 2, but the barriers differ by up to 5 kcal/mol. Combining the two sets of simulations (preliminary and additional) in the WHAM calculation of the free energy barriers gave barriers identical to those obtained for the additional simulations alone, suggesting that adequate sampling has been performed in the latter simulations. Individual barriers for each simulation were also calculated, and these are provided in the Supporting Information. The individual barriers correspond to different starting geometries, taken from the unrestrained QM/MM MD simulations. The barriers calculated from different simulations differ significantly in some cases: the highest and lowest barriers for the same process differ by as much as 10 kcal/mol and correspond to different conformational minima on the free energy surface of the protein. These findings highlight the need for adequate conformational sampling in simulations of this type, as shown previously.4 QM/MM calculations with higher levels of QM theory gave barriers that were considerably lower than those calculated at the AM1/CHARMM22 level. For example, the B3LYP/ CHARMM22 potential energy barriers were generally 10− 15 kcal/mol lower than the corresponding AM1/CHARMM22 potential energy barriers (given in the Supporting Information). However, the same reactivity trends are predicted with all levels of QM theory tested here. The free energy barriers are discussed below in two parts. First, the relative free energy barriers and experimental regioselectivity observations are compared, and second, the free energy barriers are compared with experimentally determined reaction rates for a range of EH substrates. Comparison with Experiment. Regioselectivity. The free energy barriers to nucleophilic attack on t-DPPO in the protonated model, from the preliminary and extended umbrella sampling simulations (Tables 2 and 3), show a preference for attack at C(1) over C(2). This is in qualitative agreement with the experimentally determined regioselectivity for murine sEH reported by Borhan et al., who measured a 97:3 ratio of attack at C(1) to the less-hindered C(2).22 This ratio corresponds to a difference in the barrier of ∼2 kcal/mol. This selectivity is in contrast to that observed for microsomal EH, where attack at the less hindered C(2) is preferred.26 It is encouraging to note that the regioselectivity predictions are broadly the same, regardless of the QM method used, as lower barriers for C(1) attack are observed for all of the QM methods used (see below). The preference for attack at C(1) over C(2) is observed for both 1779 dx.doi.org/10.1021/bi201722j | Biochemistry 2012, 51, 1774−1786