title: Semifunctional site-specific mutants affecting the hydrolytic half-reaction of microsomal epoxide hydrolase.
abstract: Microsomal epoxide hydrolase (MEH) is a member of the alpha/beta-hydrolase fold family of enzymes, each of which has a catalytic triad consisting of a nucleophile involved in the formation of a covalent intermediate and a general base and charge relay carboxylate that catalyze the hydrolysis of the intermediate. The rate-limiting step in the catalytic mechanism of MEH is hydrolysis of the ester intermediate. An efficient bacterial expression system for a C-terminal hexahistidine tagged version of the native enzyme, which facilitates the isolation of mutant enzymes in which residues involved in the hydrolytic half-reaction have been altered, is described. The H431S mutant of this enzyme is efficiently alkylated by substrate to form the ester intermediate but is unable to hydrolyze the ester to complete the catalytic cycle, a fact that confirms that H431 acts as the base in the hydrolytic half-reaction. The charge relay carboxylate, which is not apparent in paired sequence alignments with other alpha/beta-hydrolase fold enzymes, is thought to be located between residues 340 and 405. A mutagenic survey of all eight Asp and Glu residues in this region reveals that only two (E376 and E404) influence the catalytic mechanism. Steady-state and pre-steady-state kinetic analyses of these residues suggest that both E404 and E376 may serve the charge relay function in the hydrolysis half-reaction. Finally, the tryptophan residue (W150), which resides in the oxyanion hole sequence HGWP, is demonstrated to contribute to the large change in intrinsic protein fluorescence observed when the enzyme is alkylated.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: H F
authors.initials: HF
authors.lastname: Tzeng
authors.forename: L T
authors.initials: LT
authors.lastname: Laughlin
authors.forename: R N
authors.initials: RN
authors.lastname: Armstrong
mesh_terms: D000818:Animals; D002384:Catalysis; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D051381:Rats
chemical_list: D004851:Epoxide Hydrolases