ROLES OF SURFACE HYDROPHOBIC RESIDUES IN THE INTERFACIAL CATALYSIS OFBOVINE PANCREATIC PHOSPHOLIPASE A(2)

The interfacial binding is a unique and important step in the phosphol ipase A(2) (PLA(2)) catalyzed hydrolysis of phospholipids which is dis tinct from the binding of a substrate to the active site. To assess th e roles of surface hydrophobic residues of PLA(2) in these processes, we selectively mutated Leu-19 and Leu-20 of bovine pancreatic PLA(2) t o charged (L19K and L20K), uncharged polar (L19S and L20S), and amphip hilic (L19W and L20W) groups and measured their kinetic and binding pr operties using various phospholipid aggregates, including micelles, mo nolayers, 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 PLA(2). Toward monome ric 1,2-dihexanoyl-sn-glycero-3-phosphocholine, all Leu-20 mutants (L2 0S, 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 zwitterioni c 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC(8)PC) micelles, L20S and L20K mutants showed only 30% and 351e of the wild-type activity, respectively, whereas L20W was about twice as active as wild type. Als o, L19S and L19K showed 75% and 15% of the wild-type activity. respect ively. Toward anionic Triton X-100/sodium deoxycholate/diC(8)PC (4:2:1 ) mixed micelles, L20W and L20K were 2.6 times and twice more active t han wild type. To determine the sn-2 acyl group selectivity of wild ty pe and mutants, polymerized mixed liposomes were used which contained ipoyloxy)-dodecanoyl]-sn-glycero-3-phosphoglycerol and 1 mol % of eith er dodecanoyl]-2-hexanoyl-sn-glycero-3-phosphocholine or ecanoyl]-2-do decanoyl-sn-glycero-3-phosphocholine. These measurements showed that L eu-19 was involved in the substrate binding and the sn-2 acyl group se lectivity of bovine pancreatic PLA(2) and that Leu-20 made a direct co ntact with the surface of phospholipid aggregates. The binding affinit ies of mutants to micelles, polymerized liposomes, and monolayers were well consistent with their kinetic behaviors, supporting the notion t hat the altered activities of Leu-19 mutants and Leu-20 mutants were d ue to the change in their substrate binding and interfacial binding, r espectively. Finally, the L20W mutant represents the first example of protein engineering of PLA(2) which results in a significant increase in interfacial binding to densely packed neutral monolayers and bilaye rs.