R. Dua et al., A STRUCTURE-FUNCTION STUDY OF BOVINE PANCREATIC PHOSPHOLIPASE A(2) USING POLYMERIZED MIXED LIPOSOMES, The Journal of biological chemistry, 270(1), 1995, pp. 263-268
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.