Biochemistry carbon atoms are chemically equivalent, this is likely to be responsible for the preference for attack at C(1). To analyze the effect of the enzyme environment on the barriers, we calculated B3LYP/6-31G(d) single-point energies for the QM atoms in the absence of the MM region. This was performed on structures corresponding to the highest and lowest points on the B3LYP/CHARMM22 PESs for C(1) and C(2) nucleophilic attack for the t-SO, t-DPPO(1), and t-DPPO(2) models (with His523 protonated). The gas phase barriers were higher than those calculated in the enzyme by 3−10 kcal/mol (Table 5). This represents a rate acceleration of 102−105-fold in the enzyme, compared with that in the gas phase. Table 5. Activation Barriers (in kilocalories per mole) Calculated for Nucleophilic Attack at C(1) and C(2) in t-SO, t-DPPO(1), and t-DPPO(2) for the Protonated His523 ⧧ Model with B3LYP/6-31G(d)/CHARMM22 (ΔEQM/MM) and from Single-Point [B3LYP/6-31G(d)] Energy Calculations on the QM Atoms of the B3LYP/6-31G(d)/ CHARMM22 Structures in the Gas Phase (ΔEg⧧as) and with ⧧ Continuum Solvation (ΔEgas+solv) t-SO t-DPPO(1) t-DPPO(2) C(1) C(2) C(1) C(2) C(1) C(2) ΔEQ⧧M/MM 6.9 14.8 6.7 10.5 9.2 11.3 ΔEg⧧as 14.7 17.8 14.9 20.2 14.3 17.8 ΔEg⧧as+solv 15.6 20.5 13.0 19.2 14.0 20.2 To make the most meaningful analysis of the rate acceleration achieved by an enzyme, the activation barrier of the reaction in the enzyme should be compared to that of the same reaction in solution.41 B3LYP/6-31G(d) single-point energies were also calculated for the QM atoms in the absence of the MM region for the structures detailed above, including the Poisson−Boltzmann solvation model in Jaguar (with a dielectric constant of 80.37 and a probe radius of 1.40 Å).70 All of the barriers calculated with solvation are higher than their corresponding QM/MM barriers, by 4.8−9 kcal/mol (Table 5). For t-SO, the solvent-corrected barriers are ∼2 kcal/mol higher than the corresponding gas phase barriers, indicating a rate acceleration by the enzyme even greater than that predicted by the gas phase calculations. For t-DPPO(1) and -(2), the barriers calculated with solvation are lower than their gas phase counterparts by approximately the same amount. This might suggest that the nucleophilic attack of t-SO is more favored kinetically in a hydrophobic environment and that the reverse is true for t-DPPO. However, it is possible that the solvation effect is not significant, given the small difference in energy, which is within the expected error margins of B3LYP and con- tinuum solvent models. There are no water molecules present in the active site in the enzyme−substrate complexes in any of the QM/MM calculations, as there is insufficient space. As a water molecule is required for the hydrolysis step, it is possible that the enzyme undergoes a structural rearrangement, with the corresponding entrance of water, prior to the hydrolysis step. There are several discrepancies between the work presented here and the DFT study by Hopmann et al.50 First, our calculations indicate that the most likely protonation state of His523 is the positively charged species, whereas Hopmann et al. modeled the neutral species and do not appear to have considered the protonated model in their study. Also, their Article work is based on a small static structure of the enzyme taken from a crystal structure, whereas our modeling consists of a larger enzyme model and molecular dynamics simulations, where the mobility of amino acid residues is included. Second, Hopmann et al. suggest that the oxyanion formed after the nucleophilic attack is protonated by Tyr465. While we do not model the protonation by Tyr465 in this study, it seems unlikely to us that this step is necessary. Tyrosine is not a very strong acid, and it is likely that the proton would more readily come from the protonated His523, mediated by a water molecule. This would then leave His523 in a neutral state and allow it to activate the water molecule responsible for the hydrolysis step. This would be an interesting route for further investigation. ■ CONCLUSIONS Epoxide hydrolases have the potential to act as highly selective biocatalysts in the large-scale production of enantiopure compounds. Understanding the nature of the chemical transformations taking place in the enzyme, together with the factors that dictate the stereochemistry of the products, will lead to a better realization of their power for synthetic purposes. The nucleophilic epoxide ring opening by Asp333 has been studied here for two different epoxides: t-SO and t-DPPO. These similar epoxides differ in that the two epoxide carbon atoms are equivalent in t-SO but are not in t-DPPO. A combination of QM(AM1)/MM(CHARMM) umbrella sam- pling MD simulations, B3LYP/CHARMM22, and SCS-MP2 potential energy calculations has been used to obtain free energy barriers to nucleophilic attack at both epoxide carbon atoms in the two substrates. For t-DPPO, the nucleophilic attack was modeled for two binding orientations, placing one of the two epoxide carbon atoms closer to the nucleophile. In both cases, the nucleophilic attack at the phenyl epoxide carbon atom was found to be preferred over attack at the benzylic carbon atom. This observation is in agreement with experimental studies.22 Our AM1/CHARMM22 umbrella sampling MD simulations give relative free energy barriers that are in good agreement with experimental regioselectivity data for t-DPPO. While the difference between barriers is in good agreement, the absolute values of the free energy barriers calculated at this level of theory are too high, because of the limitations of the AM1 method. Corrections from potential energy barriers calculated with higher levels of QM theory give free energy barriers that are closer to experiment. While the barriers calculated at the B3LYP/CHARMM22 QM/MM level are somewhat too low, correction with SCS-MP2 results in free energy barriers that quantitatively agree with the range of values derived from experimental rate constants. Our work suggests that the regioselectivity of nucleophilic attack for t-DPPO in sEH is mostly due to the relative electrophilicity of the two epoxide carbon atoms. In the t-SO calculations, despite the chemical equivalence of the two epoxide carbon atoms, a preference for attack at the carbon atom closer to the nucleophile in the enzyme−substrate complex was observed. In the case of t-DPPO, the proximity of the nucleophile appears to have a weaker effect on the predicted regioselectivity, compared with electronic factors. This observation may be important in the design of epoxide hydrolase enzymes for the catalysis of specific transformations. The results indicate that simple models based purely on geometric factors37 are unlikely to be generally successful in predicting regioselectivity. 1783 dx.doi.org/10.1021/bi201722j | Biochemistry 2012, 51, 1774−1786