Lindberg_2010_Biochemistry_49_2297

The underlying enzyme kinetics behind the regioselective promiscuity shown by epoxide hydrolases toward certain epoxides has been studied. The effects of temperature and pH on regioselectivity were investigated by analyzing the stereochemistry of hydrolysis products of (1R,2R)-trans-2-methylstyrene oxide between 14-46 degrees C and pH 6.0-9.0, either catalyzed by the potato epoxide hydrolase StEH1 or in the absence of enzyme. In the enzyme-catalyzed reaction, a switch of preferred epoxide carbon that is subjected to nucleophilic attack is observed at pH values above 8. The enzyme also displays cooperativity in substrate saturation plots when assayed at temperatures < or = 30 degrees C and at intermediate pH. The cooperativity is lost at higher assay temperatures. Cooperativity can originate from a kinetic mechanism involving hysteresis and will be dependent on the relationship between k(cat) and the rate of interconversion between two different Michaelis complexes. In the case of the studied reactions, the proposed different Michaelis complexes are enzyme-substrate complexes in which the epoxide substrate is bound in different binding modes, allowing for separate pathways toward product formation. The assumption of separated, but interacting, reaction pathways is supported by that formation of the two product enantiomers also displays distinct pH dependencies of k(cat)/K(M). The thermodynamic parameters describing the differences in activation enthalpy and entropy suggest that (1) regioselectivity is primarily dictated by differences in activation entropy with positive values of both DeltaDeltaH(++) and DeltaDeltaS(++) and (2) the hysteretic behavior is linked to an interconversion between Michaelis complexes with rates increasing with temperature. From the collected data, we propose that hysteresis, regioselectivity, and, when applicable, hysteretic cooperativity are closely linked properties, explained by the kinetic mechanism earlier introduced by our group.