Substrate-dependent hysteretic behavior in StEH1-catalyzed hydrolysis of styrene oxide derivatives.
The substrate selectivity and enantioselectivity of Solanum tuberosum epoxide hydrolase 1 (StEH1) have been explored by steady-state and pre-steady-state measurements on a series of styrene oxide derivatives. A preference for the (S)- or (S,S)-enantiomers of styrene oxide, 2-methylstyrene oxide and trans-stilbene oxide was established, with E-values of 43, 160 and 2.9, respectively. Monitoring of the pre-steady-state phase of the reaction with (S,S)-2-methylstyrene oxide revealed two observed rates for alkylenzyme formation. The slower of these rates showed a negative substrate concentration dependence, as did the rate of alkylenzyme formation in the reaction with the (R,R)-enantiomer. Such kinetic behavior is indicative of an additional, off-pathway step in the mechanism, referred to as hysteresis. On the basis of these data, a kinetic mechanism that explains the kinetic behavior with all tested substrates transformed by this enzyme is proposed. Regioselectivity of StEH1 in the catalyzed hydrolysis of 2-methylstyrene oxide was determined by (13)C-NMR spectroscopy of (18)O-labeled diol products. The (S,S)-enantiomer is attacked exclusively at the C-1 epoxide carbon, whereas the (R,R)-enantiomer is attacked at either position at a ratio of 65 : 35 in favor of the C-1 carbon. On the basis of the results, we conclude that differences in efficiency in stabilization of the alkylenzyme intermediates by StEH1 are important for enantioselectivity with styrene oxide or trans-stilbene oxide as substrate. With 2-methylstyrene oxide, slow conformational changes in the enzyme also influence the catalytic efficiency.
Lindberg
Diana
D
Department of Biochemistry and Organic Chemistry, Uppsala University, Sweden.
Gogoll
Adolf
A
Widersten
Mikael
M
D021301:Antifreeze Proteins / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D013379:Substrate Specificity; D013816:Thermodynamics
D021301:Antifreeze Proteins; D004852:Epoxy Compounds; C013690:styrene oxide; D004851:Epoxide Hydrolases