title: Identification and designing of the S3 site of aqualysin I, a thermophilic subtilisin-related serine protease.
abstract: Aqualysin I is a bacterial subtilisin-related alkaline serine protease, originating in Thermus aquaticus YT-1. Based on computational analysis, we predicted that two residues, Ser102 and Gly131, form the S3 site of aqualysin I, and we proved that this prediction by site-directed mutagenesis. To alter the P3-specificity of the enzyme, we built a "wall" on the S3 site edge by introducing a bulky side chain at target sites. Six mutant proteins were prepared: S102H, S102K, S102E, G131H, G131K, and G131D. The mutant enzymes were examined with two kinetically typical peptides for aqualysin I, suc-X-Ala-Ala-pNA, where X is Ala or Phe. All mutations reduced the efficiency for the Phe-containing peptide, while they raised the k(cat) values for the Ala-containing peptide. Especially, the S102K mutant protein hydrolyzed the polyalanine peptide efficiently. The strategies we have adopted in this paper are applicable to all subtilisin-related enzymes.
authors.affiliation: Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, 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: D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D015195:Drug Design; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013824:Thermus / Q000201:enzymology / Q000235:genetics
chemical_list: D017931:DNA Primers; D009842:Oligopeptides; D012697:Serine Endopeptidases; D013381:Subtilisins; C055102:aqualysin I