High-Affinity Fluorogenic Substrate for Tissue Transglutaminase Reveals Enzymatic Hysteresis.

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Transglutaminases (TGases) are calcium-dependent enzymes catalyzing protein cross-linking.
• Transglutaminase 2 (TG2) exhibits dual functionality as a transamidase and a G-protein, regulated by stimuli and redox reactions.
• TG2's ubiquitous expression and involvement in diseases necessitate robust activity monitoring methods.

Purpose of the Study:

• To design, synthesize, and evaluate a novel substrate for monitoring Transglutaminase 2 (TG2) activity.
• To characterize the substrate's affinity, selectivity, and reactivity with TG2.
• To investigate the kinetic behavior of TG2 using the new substrate.

Main Methods:

• Design and synthesis of a TG2 activity substrate (APH7) based on an optimized inhibitor scaffold.
• Evaluation of APH7's kinetic parameters, including Michaelis constant (K M), with TG2.
• Analysis of TG2 activity and inhibition using the APH7 substrate to identify kinetic phenomena.

Main Results:

• The novel substrate APH7 demonstrates excellent affinity (K M = 3.0 μM), selectivity, and reactivity towards TG2.
• Application of APH7 enabled the discovery of unique hysteresis in TG2's catalytic and inhibitory activities.
• The substrate provides a valuable tool for studying TG2's complex enzymatic mechanisms.

Conclusions:

• APH7 is a highly effective tool for studying Transglutaminase 2 (TG2) activity and kinetics.
• The discovery of hysteresis highlights the complex regulatory mechanisms of TG2.
• This research facilitates further investigation into TG2's role in disease and therapeutic development.