Potentiometric studies on benzeneboronic acid-alpha-chymotrypsin interactions

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein chemistry

Background:

• Benzeneboronic acid (BBA) is a competitive inhibitor of alpha-chymotrypsin.
• BBA forms a complex with alpha-chymotrypsin, leading to proton release in the acidic pH range.
• Proton release is a measurable event using difference potentiometry.

Purpose of the Study:

• To propose a method for estimating equilibrium constants of BBA trigonal-tetrahedral interconversion.
• To determine thermodynamic parameters of His 57 ionization and BBA binding steps.
• To investigate the mechanism of proton release during BBA-enzyme complex formation.

Main Methods:

• Difference potentiometric technique to measure proton release.
• Temperature dependence studies to determine thermodynamic parameters.
• Comparative analysis of proton release in various chymotrypsin forms.

Main Results:

• Proton release was observed upon BBA complex formation with alpha-chymotrypsin and chymotrypsinogen A, but not with TRCK-, DIP-, and anhydrochymotrypsins.
• A procedure for estimating BBA interconversion equilibrium constants was developed.
• Thermodynamic parameters for His 57 ionization were found to be similar to imidazole in water.
• Enthalpic gain in the BBA-enzyme interaction was significantly reduced compared to BBA ionization in water, attributed to a shift in proton acceptor from water to imidazole.

Conclusions:

• The study provides a method to quantify BBA binding to alpha-chymotrypsin through proton release measurements.
• Thermodynamic insights into the BBA-enzyme interaction mechanism were gained.
• The reduced enthalpy suggests a favorable interaction driven by the enzyme's active site environment.