title: Significance of the conserved Tyr352 and Asp380 residues in the catalytic activity of Bacillus stearothermophilus aminopeptidase II as evaluated by site-directed mutagenesis.
abstract: The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.5-fold. Detailed analysis of the kinetic consequences in the mutant proteins revealed that the K (m) values were increased 1.9- to 2.6-fold with respect to wild-type enzyme. Catalytic efficiencies (k (cat)/K (m)) of mutant proteins were between 3.5- and 31-fold lower than the corresponding value of the wild-type enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results indicate that residues Tyr352 and Asp380 are essential for the proper function of AP-II.
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi 60083, Taiwan. llin@mail.ncyu.edu.tw
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
authors.forename: Yi-Pu
authors.initials: YP
authors.lastname: Chen
authors.forename: Jia-Ci
authors.initials: JC
authors.lastname: Yang
authors.forename: Yu-Wen
authors.initials: YW
authors.lastname: Hua
authors.forename: Wen-Ching
authors.initials: WC
authors.lastname: Wang
authors.forename: Lih-Ying
authors.initials: LY
authors.lastname: Kuo
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000502:physiology*; D002384:Catalysis; D002942:Circular Dichroism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D016415:Sequence Alignment; D014443:Tyrosine / Q000502:physiology*
chemical_list: D001224:Aspartic Acid; D014443:Tyrosine; D000626:Aminopeptidases