title: Chlorophyllase as a serine hydrolase: identification of a putative catalytic triad.
abstract: Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.
authors.affiliation: Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Kanagawa, 226-8501 Japan. ttsuchiy@bio.titech.ac.jp
authors.forename: Tohru
authors.initials: T
authors.lastname: Tsuchiya
authors.forename: Takuo
authors.initials: T
authors.lastname: Suzuki
authors.forename: Takafumi
authors.initials: T
authors.lastname: Yamada
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shimada
authors.forename: Tatsuru
authors.initials: T
authors.lastname: Masuda
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Ohta
authors.forename: Ken-ichiro
authors.initials: K
authors.lastname: Takamiya
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D027463:Chenopodium album / Q000201:enzymology / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006639:Histidine / Q000235:genetics; D006868:Hydrolysis; D007531:Isoflurophate / Q000494:pharmacology; D008049:Lipase / Q000378:metabolism; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics; D012697:Serine Endopeptidases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D009025:Morpholines; D007531:Isoflurophate; C008550:morpholinopropane sulfonic acid; D001224:Aspartic Acid; D012694:Serine; D006639:Histidine; D010664:Phenylmethylsulfonyl Fluoride; D020245:p-Chloromercuribenzoic Acid; D002265:Carboxylic Ester Hydrolases; C019875:chlorophyllase; D008049:Lipase; D012697:Serine Endopeptidases