Photo-regulation of hydrolysis activity of semisynthetic mutant phospholipases A2 replaced by non-natural aromatic amino acids

Abstract

Trp³ of phospholipase A₂(PLA2) was replaced by non-natural aromatic amino acids, 3-(2-naphthyl)-L-alanine (Nap), 3-(9-anthryl)-DL-alanine (Ant), and p-phenylazo-L-phenylalanine (AzoF), by a semisynthetic method. A fully ε-amidinated PLA2 (AMPA) was subjected to three cycles of Edman degradation to obtain des-Ala¹, Leu², Trp³-AMPA [des-(Ala¹-Trp³)-AMPA](DES3). Subsequently, the tripeptide having a non-natural amino acid at the third position was connected to DES3 to obtain [Nap³]AMPA (Nap-AMPA), [Ant³]AMPA (Ant-AMPA), and [AzoF³]AMPA (AzoF-AMPA). Nap-AMPA and Ant-AMPA partially retained hydrolysis activity for phospholipid membranes, while AzoF-AMPA having a trans(E) configuration of the AzoF unit lost this activity. UV irradiation during the hydrolysis reduced the activities of Nap-AMPA and Ant-AMPA. However, AzoF-AMPA with a cis(Z) configuration of the AzoF unit showed, on UV irradiation, hydrolytic activity. The change of enzymic activity induced by UV irradiation is ascribed to a conformational change of the mutant proteins.