Reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor-V: function, thermodynamic stability, and NMR solution...

Area of Science:

• Biochemistry
• Structural Biology
• Proteomics

Background:

• Cucurbita maxima trypsin inhibitor-V (CMTI-V) is a potent protease inhibitor.
• Hydrolysis of the reactive-site peptide bond (Lys44-Asp45) yields CMTI-V*.
• Understanding structural and functional changes in CMTI-V* is crucial for inhibitor design.

Purpose of the Study:

• To characterize the functional and thermodynamic properties of hydrolyzed CMTI-V* (CMTI-V*).
• To determine the three-dimensional solution structure of CMTI-V*.
• To elucidate the molecular basis for altered inhibitory activity.

Main Methods:

• Preparation and characterization of CMTI-V*.
• Enzyme inhibition assays against trypsin and human blood coagulation factor XIIa.
• Thermodynamic analysis of hydrolysis using equilibrium constants and temperature dependence.
• Nuclear Magnetic Resonance (NMR) spectroscopy for structural determination.
• Distance geometry and simulated annealing for structure computation.

Main Results:

• CMTI-V* exhibited significantly reduced inhibitory properties and binding affinities compared to intact CMTI-V.
• The equilibrium constant for trypsin-catalyzed hydrolysis (Khyd) was approximately 9.4 at 25°C.
• Thermodynamic parameters (ΔH°, ΔS°) for hydrolysis were estimated.
• The 3D solution structure of CMTI-V* was determined using NMR, yielding 20 computed structures with low root-mean-square deviation.

Conclusions:

• Hydrolysis of the reactive-site peptide bond in CMTI-V leads to a loss of inhibitory function.
• Structural and thermodynamic analyses provide insights into the molecular mechanisms underlying these functional changes.
• The findings contribute to understanding protease-inhibitor interactions and designing novel inhibitors.