Site-directed mutagenesis study of phospholipase A(2) (PLA(2)) from the pyloric ceca of starfish Asterina pectillifera was used to probe the relationship between polar-group specificity and structure of the pancreatic loop region. The sequence of the cDNA encoding the starfish PLA(2) was exchanged by the oligonucleotide-directed dual amber-long and accurate polymerase chain reaction method to insert Lys residue between Cys-62 and Gly-63. The modified cDNA was inserted into the expression plasmid pET-16b, and PLA(2) mutant was expressed in Escherichia coli Origami (TM) B (DE3) by induction with isopropyl-beta-D(-)-thiogalactopyi-anoside. The starfish PLA(2) mutant showed essentially the same properties as the starfish native PLA(2) with respect to substrate positional specificity, optimum pH, optimum temperature, Ca2+ requirement, and sodium deoxycholate requirement. However, the specific activity of the starfish PLA(2) mutant for egg yolk PC (950 U/mg) was extremely lower than that of native PLA(2) (119,000 U/mg), but close to that of porcine pancreatic PLA(2) (4300 U/mg). Moreover, the ratio of specific activity of the PLA(2) mutant for phosphatidylcholine to phosphatidylethanolamine (98 times) was highly lower than that of native PLA(2) (2650 times), but similar to that of porcine pancreatic PLA(2) (25 times). Therefore, it was suggested that the charge and structure of pancreatic loop region of the starfish PLA(2) might carry out important role on polar-group specificity. (c) 2006 Elsevier Inc. All rights reserved.