Effects of specific fatty acid acylation of phospholipase A2 on its interfacial binding and catalysis.
Monomeric phospholipase A2 (PLA2) from the venom of Agkistrodon piscivorus piscivorus (App-D49) was treated with 3-acyloxy-4-nitrobenzoic 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-D49s, with acyl groups ranging from lauroyl to palmitoyl, spontaneously aggregated in solution. By contrast, 7,10-dioctanoyl-App-D49 existed as a monomer under the same condition. Kinetic and interfacial binding properties of diacylated enzymes indicated that they catalyzed the hydrolysis at the interface as a monomer. When compared to nonacylated App-D49, diacylated enzymes showed slightly increased activity or decreased activity toward monodispersed 1,2-dibutyryl-sn-glycero-3-phosphocholine, Triton X-100/1,2-dilauroyl-sn-glycero-3-phosphocholine mixed micelles, and small unilamellar vesicles (SUV) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Toward densely-packed liquid-crystalline phospholipid bilayers, such as large unilamellar vesicles (LUV) of POPC, however, diacylated enzymes exhibited a large increase in activity, which reacted up to 250-fold for 7,10-dilauroyl-App-D49 ((kcat/Km)app = (1.0 +/- 0.02) x 10(6) M-1 s-1). Measurements of the penetration of individual diacylated enzymes into 2-oleoyl-3-palmitoyl-sn-glycero-1-phosphocholine (i.e., D-POPC) monolayers indicated that the acyl groups enhanced the interfacial binding of protein by interacting with hydrocarbon moieties of phospholipids and that these hydrophobic interactions remained effective even when the phospholipid packing density was high. Furthermore, fluorometric measurements of the binding of diacylated enzymes to polymerized vesicles of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine showed that the hydrophobic interactions increased the enzymatic activity toward LUV by accelerating the migration of enzyme molecules to vesicle surfaces. The analysis of the kinetic 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 turnovers 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 active form toward densely-packed liquid-crystalline phospholipid bilayers, which might have potential industrial and biomedical applications.
Shen
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Department of Chemistry (M/C 111), University of Illinois at Chicago 60607-7061.
Wu
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SK
Cho
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D000215:Acylation; D017836:Agkistrodon; D000818:Animals; D002384:Catalysis; D003435:Crotalid Venoms / Q000201:enzymology*; D004789:Enzyme Activation; D005227:Fatty Acids / Q000737:chemistry; D008081:Liposomes; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D013499:Surface Properties; D013816:Thermodynamics
D003435:Crotalid Venoms; D005227:Fatty Acids; D008081:Liposomes; D010741:Phospholipases A; D054467:Phospholipases A2