title: Contribution of conserved Asn residues to the inhibitory activities of Kunitz-type protease inhibitors from plants.
abstract: Plant Kunitz-type protease inhibitors contain a conserved Asn residue in the N-terminal region. To investigate the role of Asn residue in protease inhibitory activities, Erythrina variegata trypsin inhibitor a (ETIa), E. variegata chymotrypsin inhibitor (ECI), and their mutants, ETIa-N12A and ECI-N13A, were used. Both mutants exhibit weaker inhibitory activities toward their cognate proteases than the wild-type proteins and were readily cleaved at reactive sites. Furthermore, kinetic analysis of the interactions of the mutated proteins with their cognate proteases by surface plasmon resonance (SPR) measurement indicated that replacements of the Asn residue mainly affected dissociation rate constants. The conserved Asn residues of Kunitz-type inhibitors play an important role in exhibiting effective inhibitory activity by stabilizing the structures of the primary binding loop and protease-inhibitor complex.
authors.affiliation: Laboratory of Biochemistry, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
authors.forename: Shiroh
authors.initials: S
authors.lastname: Iwanaga
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Yamasaki
authors.forename: Makoto
authors.initials: M
authors.lastname: Kimura
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kouzuma
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine; D017124:Conserved Sequence / Q000235:genetics*; D004895:Erythrina / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D030781:Organisms, Genetically Modified; D010455:Peptides / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D011480:Protease Inhibitors; D001216:Asparagine