Biochemistry Scheme 1. Proposed Mechanism31,33 for Hydrolysis of Epoxide RCHOCHR′ by sEH Article Figure 1. Chemical structures of (S,S)-trans-stilbene oxide (t-SO) and (S,S)-trans-diphenylpropene oxide (t-DPPO), the substrates modeled here. The numerals 1 and 2 represent the two epoxide carbon atoms on each substrate at which nucleophilic ring opening by Asp333 can be initiated. has been observed for the regioselectivity of nucleophilic attack of (R,R)-t-MSO in StEH1, revealing interconversion between two different Michaelis complexes for this particular EH.28 Engineered mutations in StEH1 have also been used to alter the regioselectivity for nucleophilic attack by this enzyme.29,30 Understanding the causes of regioselectivity in sEH is potentially important in drug development as well as for the application of EH enzymes in biocatalysis. Two tyrosine residues (Tyr381 and Tyr465 in murine sEH) are found in the active site of all known EH enyzmes and are believed to be important in catalysis.33−36 One of these tyrosine residues has been proposed to act as a general acid catalyst in the ring-opening reaction, while the other forms a hydrogen bond to the epoxide oxygen atom in the substrate.33−36 Mutation of both of these tyrosine residues to phenylalanine leads to a 90% loss of reactivity.36 Mutation studies of EH from Agrobacterium radiobacter AD134 suggest that the residue equivalent to Tyr465 is important for the regioselectivity of the ring-opening reaction. MD simulations of sEH suggested that a so-called “near-attack conformation” (NAC, involving the nucleophilic Asp333 carboylate oxygen atom and the benzylic carbon atom of t-MSO) is populated 4 times more frequently when the epoxide oxygen atom forms a hydrogen bond to the Tyr465 side chain.37 While superficially attractive, the NAC idea suffers from problems such as the lack of a unique definition;38 also, studying reactant complexes alone can lead to misleading conclusions about enzyme reactivity.39 In general, it is important to study reactions to draw reliable conclusions. An experimental study of the hydrolysis of trans-stilbene oxide by S. tuberosum epoxide hydrolase 1 (StEH1) suggested that Tyr381 and Tyr465 act as electrophilic catalysts and do not provide a proton to the epoxide oxyanion.32 A similar role has been observed for Tyr115 in the epoxide ring-opening step in glutathione S-transferase M1-1.40 Regardless of whether they act as proton donors, it is widely accepted that Tyr381 and Tyr465 play an important role in the stabilization of the transition state and ring-opened intermediate. In general, reliable conclusions about determinants of selectivity will require the calculation of activation energy barriers for enzyme-catalyzed reactions, including thorough consideration of conformational sampling and reliable quantum chemical (or empirical valence bond41) methods.4,42 Possible causes of regioselectivity in an enzyme include intrinsic reactivity, binding mode, and specific stabilization of key species in the enzyme. QM/MM modeling is well-suited to the analysis of factors involved in regio- and stereoselectivity in enzymes, as demonstrated in a recent study of a cytochrome P450 enzyme;4 in that work, conformational sampling was found to be important in making accurate predictions of selectivity. QM/MM methods have also been used to identify a single amino acid as the likely key determinant of selectivity observed in the epoxide ring opening of phenanthrene 9,10- oxide by a glutathione S-transferase (GST).40 As with EH, the ring-opening reaction catalyzed by GST involves initial nucleophilic attack at one of the epoxide carbon atoms. In contrast to EH, in which an aspartate carboxylate oxygen atom performs this attack, in GST enzymes the nucleophilic attack is conducted by the negatively charged sulfur atom of the glutathione anion.40 In the case of transition metal-containing enzymes, such as the cytochrome P450 enzymes, consideration of conformational sampling is limited to calculating a few adiabatic mapping pathways, starting from different snapshots taken from molecular dynamics simulations.4 This is due to the poor treatment of transition metals by most semiempirical methods. For enzymes such as sEH, with no active site transition metal, semiempirical QM methods can be used to model the active site chemistry, allowing the use of sampling methods, such as MD, for generation of free energy barriers. An important question relating to the mechanism of sEH, which has yet to be answered conclusively, is whether the Nε atom of His523 (corresponding to the residue numbering in murine sEH) is protonated or unprotonated in the resting state of the enzyme, under normal conditions. This residue forms part of the catalytic triad that is mentioned above, and mutagenesis studies of both murine sEH43 and potato StEH144 have revealed that mutation of this residue results in a drastic loss of catalytic activity. The pKa of a typical imidazole side chain of a histidine residue is 6.0, whereas the estimated pKa of this residue in the murine sEH crystal structure [Protein Data Bank (PDB) entry 1CQZ] is 8.4 (calculated with PROPKA45). Microscopic and semimacroscopic methods are available for the calculation of pKa values of titratable amino acid residues in proteins;46 however (to the best of our knowledge), these have not been applied to sEH. The optimal pH range from 6.5 to 7.4 for 1775 dx.doi.org/10.1021/bi201722j | Biochemistry 2012, 51, 1774−1786