title: Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site-directed mutagenesis.
abstract: A lipase from Pseudomonas sp. MIS38 (PML) is a member of the lipase family I.3. We analyzed the roles of the five histidine residues (His(30), His(274), His(291), His(313), and His(365)) and five acidic amino acid residues (Glu(253), Asp(255), Asp(262), Asp(275), and Asp(290)), which are fully conserved in the amino acid sequences of family I.3 lipases, by site-directed mutagenesis. We showed that the mutation of His(313) or Asp(255) to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far- and near-UV circular dichroism spectra suggests that inactivation by the mutation of His(313) or Asp(255) is not due to marked changes in the tertiary structure. We propose that His(313) and Asp(255), together with Ser(207), form a catalytic triad in PML.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: H J
authors.initials: HJ
authors.lastname: Kwon
authors.forename: K
authors.initials: K
authors.lastname: Amada
authors.forename: M
authors.initials: M
authors.lastname: Haruki
authors.forename: M
authors.initials: M
authors.lastname: Morikawa
authors.forename: S
authors.initials: S
authors.lastname: Kanaya
authors.forename: K
authors.initials: K
authors.lastname: Hyun-Ju
mesh_terms: D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D004950:Esterases / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics*; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics
chemical_list: D001224:Aspartic Acid; D006639:Histidine; D004950:Esterases; D008049:Lipase