title: Studies on the molecular docking and amino Acid residues involving in recognition of substrate in proline iminopeptidase by site-directed mutagenesis.
abstract: The proline iminopeptidase (PchPiPA) of Phanerochaete chrysosporium catalyze specifically hydrolysis of N-terminal proline from peptides. The substrate Pro-pNA was docked into the catalytic pocket and several amino acid residues were identified to interact or associate with the substrate. Eight residues were selected for site-directed mutagenesis. The wild-type and mutant proteins were expressed in Escherichia coli and purified. Kinetic parameters were calculated by hydrolyzing Pro-pNA for these enzymes. Substitution of two Glu residues (Glu198 and Glu227) which interact with the substrate via formation of hydrogen bond, led to deleterious effect on catalytic efficiency (k(cat)/K(m)) due to decrease of k(cat) and increase of K(m). Four Phe residues consisting of catalytic pocket and surrounding the docked substrate, were substituted with Ala, resulting in decrease in k(cat)/K(m) to various extents. Substitution of two residues (Val267 and Cys267) localized at the deep end of the catalytic pocket also yielded negative influence on the substrate hydrolysis. Besides, all the mutants except E227Q exhibited lower thermostability than the wild-type did, indicating that these mutations may modulate the local structure. In conclusion, these amino acid residues may play an important role in maintaining local environment of the impacted catalytic pocket and be involved in recognizing or positioning the substrate.
authors.affiliation: The Key Laboratory for Biological Resources and the Environment of Ministry of Education, The Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences, Sichuan University, Chengdu, 610064, Sichuan, People's Republic of China.
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Jing
authors.forename: Hong
authors.initials: H
authors.lastname: Feng
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020075:Phanerochaete / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D005656:Fungal Proteins; D011994:Recombinant Proteins; D000626:Aminopeptidases; C022450:prolyl aminopeptidase