Interaction between squash inhibitors and bovine trypsinogen

Area of Science:

• Biochemistry
• Enzymology
• Protein-ligand interactions

Background:

• Squash seed proteinase inhibitors are known to interact with serine proteases.
• Trypsinogen is the inactive zymogen precursor of trypsin, a key digestive enzyme.
• Understanding inhibitor interactions with zymogens is crucial for enzyme regulation studies.

Purpose of the Study:

• To characterize the interaction between squash seed proteinase inhibitors and bovine trypsinogen.
• To investigate the influence of specific binding sites and cofactors on this interaction.
• To compare the binding of inhibitors to trypsinogen versus active trypsin.

Main Methods:

• Determination of association constants (Ka) for inhibitor-trypsinogen complex formation.
• Investigating the effect of calcium ions (Ca2+) on binding affinity.
• Assessing the impact of Ile-Val dipeptide binding on trypsinogen's interaction with inhibitors.
• Comparing the rate of reactive site peptide bond resynthesis.

Main Results:

• Squash seed proteinase inhibitors form weak stoichiometric complexes with bovine trypsinogen (Ka ≈ 10(4) M-1).
• Binding is significantly weaker (2x10(7) times) compared to bovine beta-trypsin.
• Inhibitors with Lys at P1 show a 2.1-fold higher Ka with trypsinogen than those with Arg at P1.
• Calcium ions and Ile-Val dipeptide binding cooperatively enhance inhibitor binding to trypsinogen (3-fold and >100-fold, respectively).
• Trypsinogen resynthesizes inhibitor peptide bonds extremely slowly in the presence of Ile-Val (10^4 times slower than beta-trypsin).

Conclusions:

• The interaction between squash seed proteinase inhibitors and trypsinogen is weak but influenced by specific structural features and cofactors.
• Cooperative binding effects involving the Ca2+ site and Ile-Val binding cleft modulate inhibitor affinity for trypsinogen.
• These findings highlight distinct regulatory mechanisms for zymogen activation and inhibitor interactions compared to active enzymes.