Use of an Imperfect Neutral Diluent and Outer Vesicle Layer Scooting Mode Hydrolysis To Analyze the Interfacial Kinetics, Inhibition, and Substrate Preferences of Bee Venom Phospholipase A2

Abstract

Interfacial catalytic constants for bee venom phospholipase A₂ (bvPLA2) have been obtained for its action on vesicles of the anionic phospholipid 1,2-dimyristoylphosphatidylmethanol (DMPM) in the highly processive scooting mode. Spectroscopic measurements which directly measure transbilayer movement of membrane components show that this exchange does not occur in anionic vesicles that have undergone complete bvPLA2-catalyzed hydrolysis of all phospholipids in the outer vesicle monolayer. 3-Hexadecyl-sn-glycero-1-phosphocholine (D-LPC) is an adequate neutral diluent for bvPLA2, which is defined as an amphiphile that forms an aggregate to which enzyme binds but neutral diluent molecules bind weakly in the enzyme's active site. D-LPC has weak affinity for the active site of bvPLA2, and theory and protocols are developed that allow its use to determine equilibrium dissociation constants for competing active site ligands. Some of the properties of bvPLA2 are shared by other 14 kDa PLA2s. (1) Ca²⁺ is required for binding of ligands to the active site but not for the binding of enzyme to the interface. (2) bvPLA2 does not significantly discriminate between phospholipids with different polar head groups or acyl chains. (3) bvPLA2 does not bind to phosphatidylcholine vesicles, and binding occurs if anionic amphiphiles are present in the vesicle. Novel features of bvPLA2 include the following: (1) Neutral diluents for other 14 kDa phospholipases A₂ are not neutral diluents for bvPLA2. (2) Saturation of the active site with a variety of different ligands does not completely prevent histidine alkylation by 2-bromo-4‘-nitroacetophenone, and Ca²⁺ binding does not change the rate of histidine alkylation. Finally, the carbohydrate portion of bvPLA2 does not alter the interfacial catalytic properties of the enzyme. Kinetic analysis of bvPLA2 in the scooting mode together with previous studies with other 14 kDa PLA2s provides a paradigm for the quantitative analysis of interfacial catalysis.