title: Catalysis of potato epoxide hydrolase, StEH1.
abstract: The kinetic mechanism of epoxide hydrolase (EC 3.3.2.3) from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 micromol x min(-1) x mg(-1) with the R,R- or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower K(m) value. The pH dependences of kcat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis- and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp105, Tyr154, Tyr235 and His300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His300-->Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis).
authors.affiliation: Department of Biochemistry, Biomedical Center, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden.
authors.forename: Lisa T
authors.initials: LT
authors.lastname: Elfström
authors.forename: Mikael
authors.initials: M
authors.lastname: Widersten
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D004851:Epoxide Hydrolases