Characteristics of phospholipase A 2 mutant of the starfish Asterina pectinifera

Abstract

Site-directed mutagenesis study of phospholipase A 2 (PLA 2) from the pyloric ceca of starfish Asterina pectinifera 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™ B (DE3) by induction with isopropyl-beta- d(−)-thiogalactopyranoside. 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, Ca 2+ 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.