TY - JOUR
T1 - Characterization of low-molecular-mass trypsin isoinhibitors from oil- rape (Brassica napus var. oleifera) seed
AU - Ascenzi, Paolo
AU - Ruoppolo, Margherita
AU - Amoresano, Angela
AU - Pucci, Piero
AU - Consonni, Roberto
AU - Zetta, Lucia
AU - Pascarella, Stefano
AU - Bortolotti, Fabrizio
AU - Menegatti, Enea
PY - 1999/4/1
Y1 - 1999/4/1
N2 - A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5-oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer than the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P1-P1' reactive site bond (where residues forming the reactive site have been identified as P(n)...P1 and P1'...P(n)', where P1-P1' is the inhibitor scissile bond) has been identified at position Arg21-Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42-Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RTI-III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H-NMR data indicates the presence of ααNOEs and 3JαNH coupling constants, typical of the β- structure(s). These data suggest that the three-dimensional structure of the RTI-III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI-III isoinhibitors binding to bovine β-trypsin and bovine α- chymotrypsin are 3.3 x 109 M-1 and 2.4 x 106 M-1, respectively, at pH 8.0 and 21.0 °C. The serine proteinase: inhibitor complex formation is a pH- dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.
AB - A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5-oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer than the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P1-P1' reactive site bond (where residues forming the reactive site have been identified as P(n)...P1 and P1'...P(n)', where P1-P1' is the inhibitor scissile bond) has been identified at position Arg21-Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42-Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RTI-III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H-NMR data indicates the presence of ααNOEs and 3JαNH coupling constants, typical of the β- structure(s). These data suggest that the three-dimensional structure of the RTI-III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI-III isoinhibitors binding to bovine β-trypsin and bovine α- chymotrypsin are 3.3 x 109 M-1 and 2.4 x 106 M-1, respectively, at pH 8.0 and 21.0 °C. The serine proteinase: inhibitor complex formation is a pH- dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.
KW - H-NMR investigation
KW - Amino acid sequence determination
KW - Brassica napus var. oleifera
KW - Disulphide bridge location
KW - Inhibitory properties
KW - Oil-rape seed
KW - Trypsin inhibitors
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U2 - 10.1046/j.1432-1327.1999.00275.x
DO - 10.1046/j.1432-1327.1999.00275.x
M3 - Article
C2 - 10103060
AN - SCOPUS:0343948320
VL - 261
SP - 275
EP - 284
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
SN - 0014-2956
IS - 1
ER -