Substrate specificity for interfacial catalysis by phospholipase A2 in the scooting mode

Area of Science:

• Biochemistry
• Enzymology
• Membrane Biophysics

Background:

• Phospholipase A2 (PLA2) enzymes are crucial for phospholipid metabolism.
• Understanding PLA2 action on different lipid interfaces is key to deciphering cellular processes.
• Previous work established PLA2 hydrolysis of anionic vesicles occurs via interfacial catalysis in a 'scooting' mode.

Purpose of the Study:

• To investigate the action of pig pancreatic phospholipase A2 on anionic phospholipid vesicles and micelles.
• To elucidate the factors influencing enzyme binding and catalytic turnover at the lipid-interface.
• To compare PLA2 activity on anionic versus zwitterionic phospholipid substrates.

Main Methods:

• Enzymatic hydrolysis assays using homologous anionic phospholipids in vesicle and micelle forms.
• Analysis of enzyme kinetics, including catalytic turnover and enzyme-substrate binding affinity.
• Investigation of substrate interface properties, such as charge distribution and acyl chain characteristics.

Main Results:

• PLA2 hydrolysis of anionic vesicles proceeds in a highly processive 'scooting' interfacial catalysis mode.
• Substrate dynamics, bilayer fluidity, and acyl chain properties have minor effects on catalytic turnover.
• Interface charge perturbation significantly alters enzyme binding, while membrane-active amphiphiles affect binding but not turnover.

Conclusions:

• Enzyme-interface binding is a primary regulator of PLA2 activity, modulated by interface charge.
• Catalytic turnover by PLA2 is largely independent of substrate dynamics and physical state.
• Anionic substrates form stable micellar complexes below the critical micelle concentration, unlike zwitterionic analogs.