title: Site-directed mutagenesis of epoxide hydrolase to probe catalytic amino acid residues and reaction mechanism.
abstract: Epoxide hydrolase from Rhodococcus opacus catalyzes the stereospecific hydrolysis of cis-epoxysuccinate to L(+)-tartrate. It shows low but significant similarity to haloacid dehalogenase and haloacetate dehalogenase (16-23% identity). To identify catalytically important residues, we mutated 29 highly conserved charged and polar amino acid residues (except for one alanine). The replacement of D18, D193, R55, K164, H190, T22, Y170, N134 and A188 led to a significant loss in the enzyme activity, indicating their involvement in the catalysis. Single and multiple turnover reaction studies show that the enzyme reaction proceeded through the two-step mechanism involving the formation of a covalent intermediate. We discuss the roles of these residues and propose its possible reaction mechanism.
authors.affiliation: Institute of Biochemistry, Zhejiang University, Hangzhou, China.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Pan
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Xie
authors.forename: Wenna
authors.initials: W
authors.lastname: Bao
authors.forename: Yongqing
authors.initials: Y
authors.lastname: Cheng
authors.forename: Jianguo
authors.initials: J
authors.lastname: Zhang
authors.forename: Yongquan
authors.initials: Y
authors.lastname: Li
mesh_terms: D000596:Amino Acids; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D004851:Epoxide Hydrolases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D012240:Rhodococcus / Q000201:enzymology; D013237:Stereoisomerism
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D004851:Epoxide Hydrolases