Binding of the recombinant proteinase inhibitor eglin c, of the soybean bowman-birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to leu-proteinase, the leucine specific serine proteinase from spinach (spinacia oleracea L.) leaves: Thermodynamic study

Paolo Ascenzi, Gino Amiconi, Alessandro Ballio, Martino Bolognesi, Enea Menegatti, Hans Peter Schnebli, Patrizia Aducci

Research output: Contribution to journalArticle

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respetively) to Leuproteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21°C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from ∼6.9, in the free Leu-proteinase, to ∼5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c - Ka = 2.2 × 1011 M-1, δG°= - 64kJ/mol, δH° = + 5.9kJ/mol, and δS° = + 240J/molK; Leu-proteinase:BBI - Ka = 3.2 × 1010 M-1, δG° = - 59kJ/mol, δH°= + 8.8kJ/mol, and δS° = + 230J/molK; and Leu-proteinase:F-C - Ka = 1.1 × 106 M-1, δG°= - 34kJ/mol, δH° = + 18J/mol, and δS° = + 180J/molK (values of Ka, δG° and δS° were obtained at 21.0°C; values of δH° were temperature-independent over the range explored, i.e. between 10.0°C and 40.0°C). F-T does not inhibit Leu-proteinase up to an inhibitor concentration of 1.0 × 10-3 M, suggesting that the upper limit of Ka is 1 × 102 M-1. Considering the known molecular models, the observed binding behaviour of eglin c, BBI, F-C and F-T to Leu-proteinase has been related to the inferred stereochemistry of the enzyme/inhibitor contact region

Original languageEnglish
Pages (from-to)283-288
Number of pages6
JournalJournal of Enzyme Inhibition and Medicinal Chemistry
Volume4
Issue number4
DOIs
Publication statusPublished - 1991

Keywords

  • Chymotrypsin inhibiting fragment (of the soybean Bowman-Birk proteinase inhibitor)
  • Leu-proteinase
  • PH and temperature effects (on proteinase:inhibitor complex formation)
  • Proteinase:inhibitor complex formation
  • Recombinant proteinase inhibitor eglin c
  • Soybean Bowman-Birk proteinase inhibitor
  • Thermodynamics (of proteinase:inhibitor complex formation)
  • Trypsin inhibiting fragment (of the soybean Bowman-Birk proteinase inhibitor)

ASJC Scopus subject areas

  • Drug Discovery
  • Pharmacology
  • Molecular Medicine
  • Biochemistry

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