A structure-function study of bovine pancreatic phospholipase A2 using polymerized mixed liposomes.
A new combinatorial approach that includes the genetic variation of protein structure and the chemical modification of phospholipid structure in polymerized mixed liposomes was used to delineate the structure-function relationships in the interfacial catalysis of bovine pancreatic phospholipase A2 (PLA2). Based on previous structural and mutational studies, several bovine PLA2 mutants were generated in which a positive charge of putatively important lysyl side chains was reversed (K10E, K53E, K56E, and K116E) or neutralized (K56Q and K116Q). Kinetic parameters of bovine wild type and mutant PLA2s determined using polymerized mixed liposomes consisting of 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoethanolamine (or -phosphoglycerol) and 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol showed that Lys-53 is involved specifically in the interaction with a substrate bound in the active site. Also, these results showed that Lys-10 and Lys-116 are involved in the interaction of bovine PLA2 with anionic interfaces but not in the interaction with the active site-bound substrate. In particular, Lys-116 makes more significant contribution than Lys-10 by approximately 1.0 kcal/mol to the binding to anionic interfaces. Most importantly, Lys-56 was shown to participate in the interaction with both the active site-bound substrate and anionic interfaces. These findings establish Lys-56 and Lys-116 as essential residues for the binding of bovine pancreatic PLA2 to anionic interfaces. Lastly, our structure-function analysis based on the use of polymerized mixed liposomes was further supported by equilibrium binding measurements of these proteins using 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol polymerized liposomes and by kinetic analyses using monomeric substrates, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine and -phosphoglycerol.
Dua
R
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Department of Chemistry, University of Illinois, Chicago 60607-7061.
Wu
S K
SK
Cho
W
W
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D007700:Kinetics; D008081:Liposomes; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D013329:Structure-Activity Relationship
D008081:Liposomes; D009838:Oligodeoxyribonucleotides; D010741:Phospholipases A; D054467:Phospholipases A2