Article pubs.acs.org/biochemistry Determinants of Reactivity and Selectivity in Soluble Epoxide Hydrolase from Quantum Mechanics/Molecular Mechanics Modeling † Richard Lonsdale, Simon Hoyle, † Daniel T. Grey,† Lars Ridder,†,‡ and Adrian J. Mulholland*,† †Centre for Computational Chemistry, School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, U.K. ‡Netherlands eScience Center, Science Park 140, 1098 XG Amsterdam, The Netherlands *S Supporting Information ABSTRACT: Soluble epoxide hydrolase (sEH) is an enzyme involved in drug metabolism that catalyzes the hydrolysis of epoxides to form their corresponding diols. sEH has a broad substrate range and shows high regio- and enantioselectivity for nucleophilic ring opening by Asp333. Epoxide hydrolases therefore have potential synthetic applications. We have used combined quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations (at the AM1/CHARMM22 level) and high-level ab initio (SCS-MP2) QM/MM calculations to analyze the reactions, and determinants of selectivity, for two substrates: trans-stilbene oxide (t-SO) and trans-diphenylpropene oxide (t-DPPO). The calculated free energy barriers from the QM/MM (AM1/CHARMM22) umbrella sampling MD simulations show a lower barrier for phenyl attack in t-DPPO, compared with that for benzylic attack, in agreement with experiment. Activation barriers in agreement with experimental rate constants are obtained only with the highest level of QM theory (SCS-MP2) used. Our results show that the selectivity of the ring-opening reaction is influenced by several factors, including proximity to the nucleophile, electronic stabilization of the transition state, and hydrogen bonding to two active site tyrosine residues. The protonation state of His523 during nucleophilic attack has also been investigated, and our results show that the protonated form is most consistent with experimental findings. The work presented here illustrates how determinants of selectivity can be identified from QM/MM simulations. These insights may also provide useful information for the design of novel catalysts for use in the synthesis of enantiopure compounds. E enzymes that catalyze the hydrolysis of epoxides to their poxide hydrolases (EHs) make up an important family of corresponding diols.1 Epoxides can be generated via endogenous biochemical pathways or, e.g., via oxidation of xenobiotics, for example, by cytochrome P450.2−4 This reaction is important in drug metabolism, as epoxides formed during the oxidation of drug molecules can react with proteins and DNA, causing damage to cells. There are five known mammalian epoxide hydrolases: microsomal and soluble epoxide hydrolases (mEH and sEH, respectively), leukotriene A4 hydrolase, cholesterol epoxide hydrolase, and hepoxilin epoxide hydro- lase.5 Although found throughout the body, sEH is highly concentrated in the kidneys and, in addition to detoxification, is involved in the metabolism of epoxyeicosatrienoic acids (EETs).2,3,6 Inhibition of sEH is a potential treatment for many diseases, including kidney failure, high blood pressure, and atherosclerosis.3,7−11 sEH has a broad substrate range, and because of its role in detoxification and biochemical modifica- tion of drugs, or their metabolites, the regioselectivity of the epoxide ring-opening reaction has important medical relevance, because of possible differences in activity between products. EHs are also used in the preparation and refinement of enantiopure diols and epoxides, which are important synthons in organic synthesis.12 Engineered EHs with designed specificities would expand their range of application considerably.13 sEH is a member of the α/β-hydrolase family. This hydrolase family shares a common catalytic triad (nucleophile-histidine- acid) and two-step mechanism, which involves the formation of a covalent ester intermediate (Scheme 1).14,15 The hydrolase activity of the enzyme is performed in the C-terminal domain, while the N-terminal domain has phosphatase activity.16−19 Previously proposed mechanisms for sEH suggested direct attack on the epoxide by an activated water molecule,20 but the mechanism involving the ester intermediate is now widely accepted.21−24 sEH can distinguish between two chemically similar epoxide carbon atoms,22 but the determinants of this regioselectivity are not known. Murine sEH preferentially attacks the intrinsically more reactive phenyl carbon of a range of aromatic substituted epoxides, including trans-diphenylpro- pene oxide (t-DPPO)22,25[i.e., C(1) in Figure 1], whereas murine mEH attacks preferentially at the less hindered benzylic epoxide carbon.26 In Solanum tuberosum epoxide hydrolase 1 (StEH1), attack at the benzylic carbon occurs exclusively for (S,S)-trans-methylstyrene oxide (t-MSO), but for (R,R)-t-MSO, the epoxide is attacked at either position, with a slight preference for the benzylic carbon.27 A temperature and pH dependence Received: November 17, 2011 Revised: January 26, 2012 Published: January 26, 2012 © 2012 American Chemical Society 1774 dx.doi.org/10.1021/bi201722j | Biochemistry 2012, 51, 1774−1786