title: Leukotriene A4 hydrolase, a bifunctional enzyme. Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297.
abstract: We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu-297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His-296, His-300 and Glu-319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site-directed mutagenesis experiments were carried out. Glu-297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu-297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Minami
authors.forename: H
authors.initials: H
authors.lastname: Bito
authors.forename: N
authors.initials: N
authors.lastname: Ohishi
authors.forename: H
authors.initials: H
authors.lastname: Tsuge
authors.forename: M
authors.initials: M
authors.lastname: Miyano
authors.forename: M
authors.initials: M
authors.lastname: Mori
authors.forename: H
authors.initials: H
authors.lastname: Wada
authors.forename: H
authors.initials: H
authors.lastname: Mutoh
authors.forename: S
authors.initials: S
authors.lastname: Shimada
authors.forename: T
authors.initials: T
authors.lastname: Izumi
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D004247:DNA; D004851:Epoxide Hydrolases / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
chemical_list: D004247:DNA; D004851:Epoxide Hydrolases; D000626:Aminopeptidases; C043134:leukotriene A4 hydrolase