Interfacial catalysis by phospholipase A2: monomeric enzyme is fully catalytically active at the bilayer interface

Area of Science:

• Biochemistry
• Enzymology
• Membrane Biophysics

Background:

• Phospholipase A2 (PLA2) enzymes play crucial roles in biological systems, including inflammation and digestion.
• Understanding the catalytic mechanism and quaternary structure of PLA2 at interfaces is essential for elucidating its function.
• Previous studies have not definitively established whether PLA2 functions as a monomer or oligomer at lipid interfaces.

Purpose of the Study:

• To investigate the interfacial catalytic activity of secretory phospholipase A2 (PLA2) from pancreatic and venom sources.
• To determine if PLA2 functions as a monomer or requires oligomerization for full catalytic activity at lipid interfaces.
• To examine the role of substrate binding and enzyme acylation in PLA2's interfacial binding and catalysis.

Main Methods:

• Enzymatic assays using 1,2-dimyristoyl-sn-glycero-3-phosphomethanol vesicles to measure hydrolysis rates.
• Varying vesicle to PLA2 ratios to analyze hydrolysis extent and initial rates under conditions minimizing molecular exchange.
• Intermolecular resonance energy transfer (RET) experiments using tryptophan and anthraniloyl fluorophores to assess enzyme proximity.

Main Results:

• PLA2 reaction progress curves showed no latency period, and hydrolysis extent correlated linearly with enzyme concentration.
• Hydrolysis per enzyme (NS) remained constant and corresponded to vesicle size, indicating monomeric activity.
• Intermolecular RET was observed only at high enzyme surface densities, supporting monomeric catalytic competence.

Conclusions:

• Secretory PLA2s from pancreas and venoms are fully catalytically active as monomers at lipid-water interfaces.
• Enzyme acylation is not required for interfacial binding or catalysis.
• Substrate binding to the active site is not a prerequisite for PLA2's interfacial adsorption.