Modifications outside the proteinase binding loop in Cucurbita maxima trypsin inhibitor III (CMTI-III) analogues change the binding energy with bovine β-trypsin

• Published in: FEBS letters 1998-10, Vol.436 (2), p.174-178
• Main Authors: Jaśkiewicz, Anna, Lis, Katarzyna, Różycki, Jan, Kupryszewski, Gotfryd, Rolka, Krzysztof, Ragnarsson, Ulf, Zbyryt, Tomasz, Wilusz, Tadeusz
• Format: Article
• Language: English
• Subjects: Acm, acetamidomethyl; Activity; Amino Acid Sequence; Amino Acid Substitution; Animals; Boc, tert-butyloxycarbonyl; Catalytic Domain; Cattle; CMTI, Cucurbita maxima trypsin inhibitor; des, removal of amino acid residue; Disulfides; E, residual enzyme concentration; HPLC, high performance liquid chromatography; I 0, initial inhibitor concentration; K a, association equilibrium constant; K m, substrate Michaelis constant; Kinetics; Models, Molecular; Molecular Sequence Data; NPGB, 4-nitrophenyl 4′-guanidinobenzoate; Peptide Fragments - chemistry; Peptides - chemical synthesis; Peptides - chemistry; Plant Proteins - chemistry; Plant Proteins - metabolism; Protein Conformation; RP, reversed phase; RT, retention time; Structure stabilization; Synthesis; Thermodynamics; Trypsin - chemistry; Trypsin - metabolism; Trypsin inhibitor; Trypsin Inhibitors - chemistry; Trypsin Inhibitors - metabolism
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)01119-3

Five 26-peptide analogues of the trypsin inhibitor [Pro 18]CMTI-III containing Leu or Tyr in position 7 and Val or Tyr in position 27: 1 (Leu 7, Tyr 27), 2 (Tyr 7, Val 27), 3 (Tyr 7, Tyr 27), 4 (Leu 7, Val 27) and 5 (Leu 7, Ala 18, Tyr 27) were synthesized by the solid-phase method. Analogues 1–4 displayed K a with bovine β-trypsin of the same order of magnitude as the wild CMTI-III inhibitor, whereas for analogue 5, this value was lower by about 3 orders of magnitude. This indicated that for the analogues with Pro (but not with Ala) in position 18, the side-chain interactions between positions 7 and 27 did not play a critical role for the stabilization of the active structure. In addition, these results also suggest that Tyr 7 is involved in an additional aromatic interaction with position 41 of the enzyme.