Site-directed mutagenesis study of starfish phospholipase A_2

摘要

Phospholipase A_2 (PLA_2, EC 3.1.1.4) from the starfish Asterinapectinifera showed high specific activity for phosphatidylcholine, compared with commercially available porcine PLA_2. In order to investigate why the starfish PLA_2 activity was so high, we expressed PLA_2 mutant (62 + K mutant) which inserted Lys residue between Cys-62 and Gly-63. The 62 + K mutant showed essentially the same enzymatic characteristics as the native PLA_2. These results indicate that the 62 + K mutant has the same three dimensional structure as that of the native PLA_2. However, the specific activities for egg yolk PC and dipalmitoyl-PC of the 62 + K mutant were extremely lower than those of the wild-type and native PLA_2. This is considered to be the result of repulsion between positively charged amino acid Lys and cationic choline base. These results suggested that the charge of pancreatic loop region of the starfish PLA_2 would play an important role in polar-group specificity.