An extended binding pocket determines the polar head group specificity of porcine pancreatic phospholipase A2.
Porcine pancreatic phospholipase A2 (PLA2) was studied by site-directed mutagenesis. Arg53 and/or Lys56 were replaced by a methionine (R53M or K56M, respectively) in combination with the Tyr69-->Phe (Y69F) substitution. These substitutions improved the activity on micellar and monomeric zwitterionic substrates and reduced the activity on negatively charged substrates compared to the Y69F mutant. With the neutral substrate 1,2-didodecanoyl-sn-glycerol-3-dimethyl phosphate (Lau2GroMe2P) a 20-fold increase of activity was observed for the 69F53M56M mutant, whereas this mutant showed a lower activity than native PLA2 on zwitterionic substrates. Thus the ratio Lau2GroMe2P/Lau2GroPCho has become 65 times higher for 69F53M56M compared to native phospholipase A2, illustrating that the substrate specificity has changed enormously. The methionine substitutions were also prepared in a 69F mutant in which a part of the surface loop (residues 62-66) was deleted. Also in this deletion mutant these substitutions showed a similar effect as the substitutions in the native 69F mutant. Furthermore it was shown that deletion of the loop increases the activity on micellar lecithins and negatively charged micellar substrates, but reduces the activity on Lau2GroMe2P. Therefore it can be concluded that the loop is important for the recognition of substrates. We also show that the loop plays a role in the dimerization of these proteins. Dimerization may account for the high activities observed for some mutants acting on monomeric substrate.
Beiboer
S H
SH
Department of Enzymology and Protein Engineering, CBLE, Utrecht University, The Netherlands.
Franken
P A
PA
Cox
R C
RC
Verheij
H M
HM
D000818:Animals; D001483:Base Sequence; D008823:Micelles; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity; D013552:Swine
D008823:Micelles; D009838:Oligodeoxyribonucleotides; D010741:Phospholipases A; D054467:Phospholipases A2