A structure-function study of bovine pancreatic phospholipase A2 using polymerized mixed liposomes

Area of Science:

• Biochemistry
• Enzymology
• Protein Structure-Function Analysis

Background:

• Bovine pancreatic phospholipase A2 (PLA2) is crucial for interfacial catalysis.
• Understanding PLA2's structure-function relationship is vital for its application.
• Previous studies identified potential roles for lysyl residues in PLA2 activity.

Purpose of the Study:

• To delineate the structure-function relationships in bovine pancreatic PLA2 interfacial catalysis.
• To investigate the roles of specific lysyl residues (Lys-10, Lys-53, Lys-56, Lys-116) in enzyme-substrate and enzyme-interface interactions.
• To utilize a combinatorial approach involving protein genetic variation and phospholipid chemical modification.

Main Methods:

• Generation of bovine PLA2 mutants by reversing or neutralizing positive charges on lysyl side chains (e.g., K10E, K53E, K56E, K116E, K56Q, K116Q).
• Kinetic parameter determination using polymerized mixed liposomes with specific phospholipid compositions.
• Supportive evidence from equilibrium binding measurements and kinetic analyses with monomeric substrates.

Main Results:

• Lys-53 is specifically involved in the interaction with an active site-bound substrate.
• Lys-10 and Lys-116 interact with anionic interfaces, with Lys-116 contributing more significantly than Lys-10.
• Lys-56 interacts with both the active site-bound substrate and anionic interfaces, highlighting its dual role.

Conclusions:

• Lys-56 and Lys-116 are essential residues for the binding of bovine pancreatic PLA2 to anionic interfaces.
• The study successfully established structure-function relationships using a novel combinatorial approach.
• Findings provide insights into the catalytic mechanism and interfacial binding of PLA2.