Interaction of peptide boronic acids with elastase: circular dichroism studies

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Serine proteases are crucial enzymes in various biological processes.
• Boronic acid derivatives of peptide substrates act as potent inhibitors.
• Slow-binding inhibition, characterized by biphasic kinetics, is a key feature of these inhibitors, suggesting they mimic reaction intermediates.

Purpose of the Study:

• To investigate the role of enzyme conformational changes in the slow-binding inhibition mechanism of pancreatic elastase by peptide boronic acids.
• To elucidate the structural basis of the biphasic binding kinetics observed with these inhibitors.

Main Methods:

• Far-ultraviolet circular dichroism (CD) spectroscopy was employed to study secondary structural changes in pancreatic elastase.
• Kinetic experiments were performed to correlate binding phases with spectral changes.
• Spectral deconvolution was used to correct for inhibitor self-assembly.

Main Results:

• No significant alterations in the secondary structure of pancreatic elastase were detected during the initial or final stages of inhibitor complex formation.
• Kinetic analysis confirmed that the slow-binding phase did not coincide with any observable CD spectral changes.
• The peptide inhibitor Ac-Pro-boro-Val-OH acted as a simple competitive inhibitor, while DNS-Ala-Pro-boro-Val-OH and Ac-Ala-Ala-Pro-boro-Val-OH exhibited slow-binding inhibition.

Conclusions:

• The slow-binding inhibition of pancreatic elastase by peptide boronic acids does not involve a significant conformational change in the enzyme's secondary structure.
• The observed biphasic kinetics are not driven by protein conformational rearrangements.
• These findings suggest alternative mechanisms for the slow-binding phenomenon in enzyme inhibition.