title: Analysis of a catalytic acidic pair in the active center of cellulase from Aspergillus aculeatus.
abstract: Four acidic amino acid residues, Asp97, Asp101, Glu118, and Glu202, were located in the cleft from the X-ray crystallographic analysis of FI-CMCase, endo-1,4-beta-glucanase (EC: 3.2.1.4) of Aspergillus aculeatus No. F-50. To identify the catalytic residues of the FI-CMCase, these residues were mutated to Glu or Ser from Asp97 and Asp101, and to Asp or Ser from Glu118 and Glu202 by site-directed mutagenesis, and totally 8 single mutant enzymes expressed in Escherichia coli were prepared: D97E, D97S, D101E, D101S, E118D, E118S, E202D, and E202S. Mutant enzymes E118S and E202S were not shown to have any detectable activity. Kinetic parameters of other mutant enzymes were measured after purification. The Km of mutant enzymes were not much different from that of wild type FI-CMCase, while the Vmax of mutant enzymes D97E, D97S, D101E, D101S, E118D, and D202E were much decreased to 1/50, 1/20, 1/4000, 1/2000, 1/800, and 1/1600 of the wild type FI-CMCase, respectively. From these results we concluded that Glu118 and Glu202 were most probable candidates for a catalytic pair of acidic amino acids in FI-CMCase.
authors.affiliation: Department of Molecular Chemistry, Graduate School of Engineering, Hokkaido University, Sapporo, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Ohnishi
authors.forename: T
authors.initials: T
authors.lastname: Ooi
authors.forename: S
authors.initials: S
authors.lastname: Kinoshita
authors.forename: H
authors.initials: H
authors.lastname: Tomatsuri
authors.forename: K
authors.initials: K
authors.lastname: Umeda
authors.forename: S
authors.initials: S
authors.lastname: Ueda
authors.forename: Y
authors.initials: Y
authors.lastname: Hata
authors.forename: M
authors.initials: M
authors.lastname: Arai
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001230:Aspergillus / Q000201:enzymology*; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D018698:Glutamic Acid / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment
chemical_list: D018698:Glutamic Acid; D002480:Cellulase