Contribution of conserved Asn residues to the inhibitory activities of Kunitz-type protease inhibitors from plants.
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.
Iwanaga
Shiroh
S
Laboratory of Biochemistry, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Yamasaki
Nobuyuki
N
Kimura
Makoto
M
Kouzuma
Yoshiaki
Y
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
C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D011480:Protease Inhibitors; D001216:Asparagine