Roles of surface hydrophobic residues in the interfacial catalysis of bovine pancreatic phospholipase A2.
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.
Lee
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Department of Chemistry (M/C 111), University of Illinois at Chicago, 60607-7061, USA.
Yoon
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Cho
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli; D007700:Kinetics; D008958:Models, Molecular; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010179:Pancreas / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000138:chemical synthesis / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013499:Surface Properties; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2; C028694:1-palmitoyl-2-oleoylphosphatidylcholine