A STRUCTURE-FUNCTION STUDY OF BOVINE PANCREATIC PHOSPHOLIPASE A(2) USING POLYMERIZED MIXED LIPOSOMES

A new combinatorial approach that includes the genetic variation of pr otein structure and the chemical modification of phospholipid structur e in polymerized mixed liposomes was used to delineate the structure-f unction relationships in the interfacial catalysis of bovine pancreati c phospholipase A(2) (PLA(2)). Based on previous structural and mutati onal studies, several bovine PLA(2) 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). Kineti c parameters of bovine wild type and mutant PLA(2)s determined using p olymerized mixed liposomes consisting of 1-pyrenedecanoyl)-sn-glycero- 3-phosphoethanolamine (or -phosphoglycerol) and lipoyloxy)dodecanoyl]- sn-glycero-3-phosphoglycerol showed that Lys-53 is involved specifical ly 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 inte raction of bovine PLA(2) with anionic interfaces but not in the intera ction with the active site-bound substrate, In particular, Lys-116 mak es more significant contribution than Lys-10 by similar to 1.0 kcal/mo l to the binding to anionic interfaces. Most importantly, Lys-56 was s hown 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 PL A(2) to anionic interfaces. Lastly, our structure-function analysis ba sed on the use of polymerized mixed liposomes was further supported by equilibrium binding measurements of these proteins using lipoyloxy)do decanoyl]-sn-glycero-3-phosphoglycerol polymerized liposomes and by ki netic analyses using monomeric substrates, 1,2-dihexanoyl-sn-glycero-3 -phosphoethanolamine and -phosphoglycerol.