Pepsinogen activation: genesis of the binding site

Area of Science:

• Biochemistry
• Enzymology
• Protein Folding

Background:

• Pepsinogen activation is a key process in gastric acid secretion.
• Previous studies suggested early formation of the substrate binding site during pepsinogen activation.

Purpose of the Study:

• To investigate the kinetics of substrate binding site appearance during pepsinogen activation.
• To determine the mechanism of pepsinogen's unimolecular activation.

Main Methods:

• Utilized a fluorescent pepstatin analogue as a probe for binding site detection.
• Employed stopped-flow fluorescence spectroscopy to monitor binding kinetics upon acidification.
• Analyzed kinetic data at various pH values.

Main Results:

• Observed a two-term exponential decay in fluorescence, indicating complex kinetics.
• Ruled out a sequential activation model.
• Demonstrated that a concurrent model with two simultaneously transforming species fits the data.
• Identified a protonation event with a pKa of approximately 2.2 influencing the two species.

Conclusions:

• Pepsinogen activation proceeds via a concurrent mechanism, not a sequential one.
• A protonation event early in activation likely induces conformational changes, leading to two distinct species.
• These conformers may persist throughout the activation process.