Roles of Surface Hydrophobic Residues in the Interfacial Catalysis of Bovine Pancreatic Phospholipase A₂

Abstract

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.

Interplay of C1 and C2 Domains of Protein Kinase C-α in Its Membrane Binding and Activation

“…Differential Roles of C1 and C2 Domains in Membrane Binding of PKC-␣-For most peripheral membrane binding proteins, both electrostatic and hydrophobic interactions play roles in their membrane binding, although their relative contributions vary with the type of proteins (17,41,42). We have shown that the membrane binding of PKC-␣ is also driven by these interactions; electrostatic interactions of the protein-bound calcium ions and other cationic residues of PKC-␣ with anionic phospholipids and hydrophobic interactions resulting from the Ca 2ϩ -and PS-dependent penetration of PKC into the hydrophobic core of the membrane (4, 12).…”

Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2) Specifically Induces Membrane Penetration and Deformation by Bin/Amphiphysin/Rvs (BAR) Domains

“…Monolayer Measurements-The penetration of PH domains into the phospholipid monolayers of different lipid compositions was measured in terms of the change in surface pressure () using a 10-ml circular Teflon trough and a Wilhelmy plate connected to a Cahn microbalance as described previously (40,(43)(44)(45)(46).…”

Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity

“…Mutants of human group IIA [41] and pancreatic group I PLA # s [42][43][44] have also shown the importance of tryptophan residues on the IBS for enhanced binding to both neutral and anionic vesicles. Of the AtxA Phe#% mutants studied in the present paper, F24W shows the highest enzymic activity, close to that of the wild type.…”