title: Epoxide hydrolase-catalyzed enantioselective conversion of trans-stilbene oxide: Insights into the reaction mechanism from steady-state and pre-steady-state enzyme kinetics.
abstract: A detailed kinetic study based on steady-state and pre-steady-state measurements is described for the highly enantioselective epoxide hydrolase Kau2. The enzyme, which is a member of the α/β-hydrolase fold family, preferentially reacts with the (S,S)-enantiomer of trans-stilbene oxide (TSO) with an E value of ∼200. The enzyme follows a classical two-step catalytic mechanism with formation of an alkyl-enzyme intermediate in the first step and hydrolysis of this intermediate in a rate-limiting second step. Tryptophan fluorescence quenching during TSO conversion appears to correlate with alkylation of the enzyme. The steady-state data are consistent with (S,S) and (R,R)-TSO being two competing substrates with marked differences in k(cat) and K(M) values. The high enantiopreference of the epoxide hydrolase is best explained by pronounced differences in the second-order alkylation rate constant (k2/K(S)) and the alkyl-enzyme hydrolysis rate k3 between the (S,S) and (R,R)-enantiomers of TSO. Our data suggest that during conversion of (S,S)-TSO the two active site tyrosines, Tyr(157) and Tyr(259), serve mainly as electrophilic catalysts in the alkylation half-reaction, polarizing the oxirane oxygen of the bound epoxide through hydrogen bond formation, however, without fully donating their hydrogens to the forming alkyl-enzyme intermediate.
authors.affiliation: Aix-Marseille Université, Centrale Marseille, CNRS, ISM2 UMR7313, Marseille 13397, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Archelas
authors.affiliation: Aix-Marseille Université, Centrale Marseille, CNRS, ISM2 UMR7313, Marseille 13397, France.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Aix-Marseille Université, Centrale Marseille, CNRS, ISM2 UMR7313, Marseille 13397, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Faure
authors.affiliation: Aix-Marseille Université, Centrale Marseille, CNRS, ISM2 UMR7313, Marseille 13397, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Iacazio
authors.affiliation: Laboratory of Biotransformation, Institute of Microbiology of the Czech Academy of Sciences, v. v. i., The Czech Academy of Sciences, Vídeňská 1083, CZ-142 20 Prague, Czech Republic. Electronic address: kotik@biomed.cas.cz.
authors.forename: Michael
authors.initials: M
authors.lastname: Kotik
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D004795:Enzyme Stability; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000648:ultrastructure*; D007700:Kinetics; D008956:Models, Chemical; D062105:Molecular Docking Simulation; D011485:Protein Binding; D011487:Protein Conformation; D013267:Stilbenes / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D013267:Stilbenes; D004851:Epoxide Hydrolases; C025906:stilbene oxide
keywords: Catalytic mechanism; Electrophilic catalysis; Enantioselectivity; Epoxide hydrolase; Fluorescence quenching; Tyrosine ionization