Human cathepsin B is a metastable enzyme stabilized by specific ionic interactions associated with the active site

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Cathepsin B is a crucial lysosomal protease involved in various cellular processes.
• Understanding the stability of cathepsin B under different conditions is vital for its study and therapeutic applications.

Purpose of the Study:

• To investigate the impact of neutral to alkaline pH on cathepsin B activity and structural integrity.
• To elucidate the relationship between pH-induced unfolding and enzyme inactivation.

Main Methods:

• Enzyme activity assays were performed at varying pH levels.
• Circular dichroism spectroscopy was used to monitor structural changes.
• Kinetic analysis was employed to determine the order and activation energy of inactivation and unfolding.

Main Results:

• Cathepsin B exhibited irreversible loss of activity and significant structural changes at pH ≥ 7.0.
• Both inactivation and unfolding followed first-order kinetics, increasing exponentially with pH.
• A high activation energy (183.5 kJ mol-1) was calculated for inactivation, consistent with observed structural alterations.
• Cathepsin B stability was compromised by increased ionic strength and organic solvent concentration.

Conclusions:

• Neutral and alkaline pH conditions lead to irreversible denaturation of cathepsin B.
• The observed correlation between unfolding and inactivation rates underscores a direct structure-function relationship.
• Cathepsin B exhibits limited stability outside of acidic environments and is sensitive to ionic strength and solvent composition.