Recent genetic and structural studies have shed considerable light on the mechanism by which secretory phospholipases A 2 interact with substrate aggregates. Electrostatic forces play an essential role in optimizing interfacial catalysis. Efficient and productive adsorption of the Class I bovine pancreatic phospholipase A 2 to anionic interfaces is dependent upon the presence of two nonconserved lysine residues at sequence positions 56 and 116, implying that critical components of the adsorption surface differ among enzyme species (Dua, R., Wu, S.-K., and Cho, W. (1995) J. Biol. Chem. 270, 263-268). In an effort to further characterize the protein residues involved in interfacial catalysis, we have determined the high resolution (1.7 Å) x-ray structure of the Class II Asp-49 phospholipase A 2 from the venom of Agkistrodon piscivorus piscivorus. Correlation of the three-dimensional coordinates with kinetic data derived from sitedirected mutations near the amino terminus (E6R, K7E, K10E, K11E, and K16E) and the active site (K54E and K69Y) defines much of the interface topography. Lysine residues at sequence positions 7 and 10 mediate the adsorption of A. p. piscivorus phospholipase A 2 to anionic interfaces but play little role in the enzyme's interaction with electrically neutral surfaces or in substrate binding. Compared to the native enzyme, the mutant proteins K7E and K10E demonstrate comparable (20-fold) decreases in affinity and catalysis on polymerized mixed liposomes of 1 -hexadecanoyl-2-(1-pyrenedecanoyl) -sn-glycero-3-phosphocholine and 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol, while the double mutant, K7E/ K10E, shows a more dramatic 500-fold decrease in catalysis and interfacial adsorption. The calculated contributions of Lys-7 and Lys-10 to the free energy of binding of A. p. piscivorus phospholipase A 2 to anionic liposomes (؊1.8 kcal/mol at 25°C per lysine) are additive (i.e. ؊3.7 kcal/mol) and together represent nearly half of the total binding energy. Although both lysine side chains lie exposed at the edge of the proposed interfacial adsorption surface, they are geographically remote from the corresponding interfacial determinants for the bovine enzyme. Our results confirm that interfacial adsorption is largely driven by electrostatic forces and demonstrate that the arrangement of the critical charges (e.g. lysines) is species-specific. This variability in the topography of the adsorption surface suggests a corresponding flexibility in the orientation of the active enzyme at the substrate interface.Phospholipases A 2 (PLA 2 ; EC 3.1. 1.4) 1 catalyze the hydrolysis of the fatty acid ester in the 2-position of 3-sn-phospholipids and are found both in intracellular and secreted forms (for recent reviews, see Refs. 1-4). The enzymes act at the lipidwater interface with a preference for organized lipids (micelles and vesicles) that is often orders of magnitudes greater than that shown for dispersed substrate. Calcium-mediated catalysis appears to involve two kinetically and struc...
Lysophosphatidic acid (LPA) is a potent lipid second messenger which stimulates platelet aggregation, cell proliferation and smooth-muscle contraction. The phospholipase A2 (PLA2)-catalysed hydrolysis of phosphatidic acid (PA) is thought to be a primary synthetic route for LPA. Of the multiple forms of PLA2 present in human tissues, human secretory class-II PLA2 (hs-PLA2) has been implicated in the production of LPA from platelets and whole blood cells challenged with inflammatory stimuli. To explore further the possibility that hs-PLA2 is involved in the production of LPA, we rigorously measured the phospholipid head group specificity of hs-PLA2 by a novel PLA2 kinetic system using polymerized mixed liposomes. Kinetic analysis of recombinant hs-PLA2 demonstrates that hs-PLA2 strongly prefers PA as substrate over other phospholipids found in the mammalian plasma membrane including phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The order of preference is PA >> PE approximately PS > PC. To identify amino acid residues of hs-PLA2 that are involved in its unique substrate specificity, we mutated two residues, Glu-56 and Lys-69, which were shown to interact with the phospholipid head group in the X-ray-crystallographic structure of the hs-PLA2-transition-state-analogue complex. The K69Y mutant showed selective inactivation toward PA whereas the E56K mutant displayed a most pronounced inactivation to PE. Thus it appears that Lys-69 is at least partially involved in the PA specificity of hs-PLA2 and Glu-56 in the distinction between PE and PC. In conjunction with a recent cell study [Fourcade, Simon, Viode, Rugani, Leballe, Ragab, Fournie, Sarda and Chap (1995) Cell 80, 919-927], these studies suggest that hs-PLA2 can rapidly hydrolyse PA molecules exposed to the outer layer of cell-derived microvesicles and thereby produce LPA.
Mammalian secretory class V phospholipase A2 (PLA2) is a newly discovered PLA2 that is implicated in eicosanoid formation in inflammatory cells. As a first step towards understanding the structure, function and regulation of this PLA2, we constructed a bacterial expression vector for human secretory class V PLA2 (hV-PLA2), over-expressed and purified the protein, and determined its physical and kinetic properties. When compared with human class IIa enzyme (hIIa-PLA2), hV-PLA2 has several distinct properties. First, hV-PLA2 can catalyse the hydrolysis of phosphatidylcholine more effectively than hIIa-PLA2 by two orders of magnitude. Secondly, hV-PLA2 has much higher binding affinity and activity for compactly packed phosphatidylcholine bilayers than hIIa-PLA2. Finally, hV-PLA2 has much reduced thermal stability compared with hIIa-PLA2. These data suggest that hV-PLA2 is better suited than hIIa-PLA2 for acting on the outer cellular membrane and liberating arachidonic acid from membrane phospholipids. Also, the unusually low thermal stability of hV-PLA2 might contribute to tighter regulation of its activities in extracellular media.
The interfacial binding is a unique and important step in the phospholipase A2 (PLA2) catalyzed hydrolysis of phospholipids which is distinct from the binding of a substrate to the active site. To assess the roles of surface hydrophobic residues of PLA2 in these processes, we selectively mutated Leu-19 and Leu-20 of bovine pancreatic PLA2 to charged (L19K and L20K), uncharged polar (L19S and L20S), and amphiphilic (L19W and L20W) groups and measured their kinetic and binding properties using various phospholipid aggregates, including micelles, monolayers, and polymerized mixed liposomes. The mutations of Leu-19 and Leu-20 did not significantly change either the tertiary structure or the thermodynamic stability of bovine pancreatic PLA2. Toward monomeric 1,2-dihexanoyl-sn-glycero-3-phosphocholine, all Leu-20 mutants (L20S, L20W, and L20K) showed activities comparable to that of wild type whereas the substitution of Leu-19 with less hydrophobic side chains (L19S and L19K) reduced the activity to 70% and 50%. Toward zwitterionic 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC8PC) micelles, L20S and L20K mutants showed only 30% and 35% of the wild-type activity, respectively, whereas L20W was about twice as active as wild type. Also, L19S and L19K showed 75% and 15% of the wild-type activity, respectively. Toward anionic Trition X-100/sodium deoxycholate/diC8PC (4:2:1) mixed micelles, L20W and L20K were 2.6 times and twice more active than wild type. To determine the sn-2 acyl group selectivity of wild type and mutants, polymerized mixed liposomes were used which contained 1,2-bis[12-(lipoyloxy)-dodecanoyl]-sn-glycero-3-phosphoglycerol and 1 mol % of either 1-2[12-(1-pyrenebutanoyloxy)dodecanoyl]-2-hexanoyl-sn-glycero-3-++ +phosphocholine or 1-[12-(1-pyrenebutanoyloxy)dodecanoyl]-2-dodecanoyl-sn-glycero-3-+ ++phosphocholine. These measurements showed that Leu-19 was involved in the substrate binding and the sn-2 acyl group selectivity of bovine pancreatic PLA2 and that Leu-20 made a direct contact with the surface of phospholipid aggregates. The binding affinities of mutants to micelles, polymerized liposomes, and monolayers were well consistent with their kinetic behaviors, supporting the notion that the altered activities of Leu-19 mutants and Leu-20 mutants were due to the change in their substrate binding and interfacial binding, respectively. Finally, the L20W mutant represents the first example of protein engineering of PLA2 which results in a significant increase in interfacial binding to densely packed neutral monolayers and bilayers.
To determine the mechanism of calcium-dependent membrane binding of cytosolic phospholipase A2 (cPLA2), we measured the interactions of cPLA2 with phospholipid monolayers and polymerizable mixed liposomes containing various phospholipids. In the presence of calcium, cPLA2 showed much higher penetrating power than secretory human pancreatic PLA2 toward anionic and electrically neutral phospholipid monolayers. cPLA2 also showed ca. 30-fold higher binding affinity for nonpolymerized 2, 3-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-1-phosphoglycerol (D-BLPG) liposomes than for polymerized ones where the membrane penetration of protein is significantly restricted. Consistent with this difference in membrane binding affinity, cPLA2 showed 20-fold higher activity toward fluorogenic substrates, 1-O-(1-pyrenedecyl)-2-arachidonoyl-sn-glycero-3-phosphocholine, inserted in nonpolymerized D-BLPG liposomes than the same substrate in polymerized D-BLPG liposomes. Furthermore, cPLA2 showed much higher sn-2 acyl group specificity (arachidonate specificity) and headgroup specificity in nonpolymerized D-BLPG liposomes than in polymerized D-BLPG liposomes. Finally, diacylglycerols, such as 1, 2-dioleoyl-sn-glycerol, selectively enhanced the membrane penetration, hydrophobic membrane binding, and interfacial enzyme activity of cPLA2. Taken together, these results indicate the following: (1) calcium not only brings cPLA2 to the membrane surface but also induces its membrane penetration. (2) This unique calcium-dependent membrane penetration of cPLA2 is necessary for its interfacial binding and substrate specificity. (3) Diacylglycerols might work as a cellular activator of cPLA2 by enhancing its membrane penetration and hydrophobic membrane binding.
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