title: Identification of glutamate residues important for catalytic activity of Bacillus stearothermophilus leucine aminopeptidase II.
abstract: Each of four conserved glutamate residues of Bacillus stearothermophilus leucine aminopeptidase II (BsLAPII) was replaced with aspartate, lysine, and leucine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes were purified to homogeneity by nickel-chelate chromatography and the molecular mass of the subunit was determined to be 44.5 kDa by SDS-PAGE. The specific activity for the Glu-316 and Glu-340 mutants was completely abolished, while Glu-249 mutants showed comparable activity to that of the wild-type BsLAPII. Compared with the wild-type enzyme, the E250D and E250L mutant enzymes retained less than 18% of the enzyme activity and exhibited a dramatic decrease in the value of k (cat)/K (m). These observations indicate that Glu-250, Glu-316, and Glu-340 residues are critical for the catalytic activity of BsLAPII.
authors.affiliation: Graduate Institute of Biotechnology, National Chiayi University, 300 University Road, 60083 Chiayi, Taiwan.
authors.forename: Hsin-Ling
authors.initials: HL
authors.lastname: Yang
authors.forename: Ruey-Shyang
authors.initials: RS
authors.lastname: Chen
authors.forename: Wenlung
authors.initials: W
authors.lastname: Chen
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D018698:Glutamic Acid / Q000032:analysis*; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D016415:Sequence Alignment
chemical_list: D021122:Protein Subunits; D018698:Glutamic Acid; D007931:Leucyl Aminopeptidase