Abstract
Isothermal titration calorimetry (ITC) technique was used to study the interactions of trypsin with bicyclic sunflower-derived trypsin inhibitor (SFTI-1) as well as with its new monocyclic (with disulphide bridge only) analogues (C3H5O)–SFTI-1 and (C8H15O)–SFTI-1. ITC measurements were run in 50 mM buffer solution of HEPES or Tricine of pH 8, containing 20 mM CaCl2 at 298.15 K. Based on calorimetric data, the equilibrium constants for the inhibitor–enzyme-binding processes, K, the binding stoichiometry, N (inhibitor-to-enzyme molar ratio), as well as thermodynamic parameters (ΔG, ΔH, ΔS) for the reactions were determined. The study revealed that the stoichiometry of the resulting complexes equals 1:1. The negative binding enthalpy (ΔITC H) and favourable entropy factor (TΔITC S) suggest an important contribution of hydrogen bonding as well as hydrophobic interactions to the inhibitor–enzyme affinity. Furthermore, the relationship between the modification of the peptide structure, the experimental conditions and the thermodynamic parameters has been discussed.
Highlights
Serine proteases are found ubiquitously in living organisms and viral genomes [1, 2]
Isothermal titration calorimetry (ITC) technique was used to study the interactions of trypsin with bicyclic sunflower-derived trypsin inhibitor (SFTI-1) as well as with its new monocyclic analogues (C3H5O)–SFTI-1 and (C8H15O)–SFTI-1
It has been found that the stoichiometry of the inhibitor–enzyme complexes equals 1:1, and their stability is governed by hydrogen bonding as well as hydrophobic interactions
Summary
Serine proteases are found ubiquitously in living organisms and viral genomes [1, 2]. The vast majority of serine proteases have chymotrypsin-like or subtilisin-like fold [3] Their name is derived from Ser195 (chymotrypsin numbering) residue found in an enzyme active site. The Ser c-hydroxyl group is a nucleophile that attacks a carbonyl group of the backbone of peptidic substrate and forms a tetrahedral intermediate. This action is facilitated by His residue acting here as a general base. The covalent acyl-enzyme complex is attacked by water molecule, leading to the formation of a new tetrahedral intermediate that undergoes a subsequent breakdown. A second peptide product is released, and the enzyme Ser195 is regenerated
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
