title: Engineering of S2 site of aqualysin I; alteration of P2 specificity by excluding P2 side chain.
abstract: Gly101, one of the conserved amino acid residues which was expected to be comprised in half-sphere-shaped S2 site small pocket of aqualysin I, a microbial thermophilic alkaline serine protease, was replaced by alanine, valine, or leucine to alterate the P2 specificity of the enzyme by excluding bulky P2 side chain of the substrate. By the mutation of G101A, the catalytic efficiencies of the enzyme for bulky amino acid residues in P2 site such as valine and leucine drastically decreased by excluding the P2 side chain. By the mutation of G101V, even the side chain of the methyl group of the alanine and the side chain of proline were excluded, while the catalytic efficiency toward glycine residue was retained. The enzyme was altered to be glycine preferable. The mutation of G101L reduced catalytic efficiencies for any substrate including glycine which is corresponding to the main chain of the peptide substrate. The strategies we have adopted in this paper are applicable to all subtilisin-related enzymes.
authors.affiliation: Department of Biotechnology, The University of Tokyo, Japan. tanakat@eco.tut.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Tanaka
authors.forename: H
authors.initials: H
authors.lastname: Matsuzawa
authors.forename: T
authors.initials: T
authors.lastname: Ohta
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D003198:Computer Simulation; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011392:Proline; D012697:Serine Endopeptidases; C055102:aqualysin I; D000409:Alanine