EFFECTS OF SPECIFIC FATTY-ACID ACYLATION OF PHOSPHOLIPASE A(2) ON ITSINTERFACIAL BINDING AND CATALYSIS

Monomeric phospholipase A(2) (PLA(2)) from the venom of Agkistrodon pi scivorus piscivorus (App-D49) was treated with 3-acyloxy-4-nitrobenzoi c acids to acylate the epsilon-amino groups of two lysines (Lys-7 and Lys-10) in the amino terminal region. Resulting 7,10-diacylated-App-D4 9s, with acyl groups ranging from lauroyl to palmitoyl, spontaneously aggregated in solution. By contrast, 7,10-dioctanoyl-App-D49 existed a s a monomer under the same condition. Kinetic and interfacial binding properties of diacylated enzymes indicated that they catalyzed the hyd rolysis at the interface as a monomer. When compared to nonacylated Ap p-D49, diacylated enzymes showed slightly increased activity or decrea sed activity toward monodispersed 1,2-dibutyryl-sn-glycero-3-phosphoch olin Triton X-100/1,2-dilauroyl-sn-glycero-3-phosphocholine mixed mice lles, and small unilamellar vesicles (SUV) of 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphochorine (POPC). Toward densely-packed liquid-crystall ine phospholipid bilayers, such as large unilamellar vesicles (LUV) of POPC, however, diacylated enzymes exhibited a large increase in activ ity, which reached up to 250-fold for 7,10-dilauroyl-App-D49 ((k(cat)/ K-m)(app) = (1.0 +/- 0.02) x 10(6) M(-1) s(-1)). Measurements of the p enetration of individual diacylated enzymes into 2-oleoyl-3-palmitoyl- sn-glycero-1-phosphochorine (i.e., D-POPC) monolayers indicated that t he acyl groups enhanced the interfacial binding of protein by interact ing with hydrocarbon moieties of phospholipids and that these hydropho bic interactions remained effective even when the phospholipid packing density was high. Furthermore, fluorometric measurements of the bindi ng of diacylated enzymes to polymerized vesicles of (lipoyloxy)dodecan oyl]-sn-glycero-3-phosphocholine showed that the hydrophobic interacti ons increased the enzymatic activity toward LUV by accelerating the mi gration of enzyme molecules to vesicle surfaces. The analysis of the k inetic course of POPC LUV hydrolysis showed that diacylated enzymes as a catalyst were superior to nonacylated App-D49 in that they were not only more catalytically efficient but also able to catalyze more turn overs without being trapped in product-containing vesicles. In summary , the acylation of App-D49 by 3-acyloxy-4-nitrobenzoic acids provides a simple and convenient way of converting the enzyme into a highly act ive form toward densely-packed liquid-crystalline phospholipid bilayer s, which might have potential industrial and biomedical applications.