The Chemistry of Escapin: Identification and Quantification of the Components in the Complex Mixture Generated by an L-Amino Acid Oxidase in the Defensive Secretion of the Sea Snail Aplysia californica

Abstract

A complex mixture of products in an enzymatic reaction: Aplysia californica releases amino acid oxidase and its substrate lysine in defensive secretions to produce a mixture of multiple compounds (see figure).

Escapin is an L-amino acid oxidase in the ink of a marine snail, the sea hare Aplysia californica, which oxidizes L-lysine (1) to produce a mixture of chemicals which is antipredatory and antimicrobial. The goal of our study was to determine the identity and relative abundance of the constituents of this mixture, using molecules generated enzymatically with escapin and also using products of organic syntheses. We examined this mixture under the natural range of pH values for ink—from ≈5 at full strength to ≈8 when fully diluted in sea water. The enzymatic reaction likely forms an equilibrium mixture containing the linear form α-keto-ε-aminocaproic acid (2), the cyclic imine Δ¹-piperidine-2-carboxylic acid (3), the cyclic enamine Δ²-piperidine-2-carboxylic acid (4), possibly the linear enol 6-amino-2-hydroxy-hex-2-enoic acid (7), the α-dihydroxy acid 6-amino-2,2-dihydroxy-hexanoic acid (8), and the cyclic aminol 2-hydroxy-piperidine-2-carboxylic acid (9). Using NMR and mass spectroscopy, we show that 3 is the major component of this enzymatic product at any pH, but at more basic conditions, the equilibrium shifts to produce relatively more 4, and at acidic conditions, the equilibrium shifts to produce relatively more 2, 7, and/or 9. Studies of escapin’s enzyme kinetics demonstrate that because of the high concentrations of escapin and L-lysine in the ink secretion, millimolar concentrations of 3, H₂O_2, and ammonia are produced, and also lower concentrations of 2, 4, 7, and 9 as a result. We also show that reactions of this mixture with H₂O₂ produce δ-aminovaleric acid (5) and δ-valerolactam (6), with 6 being the dominant component under the naturally acidic conditions of ink. Thus, the product of escapin’s action on L-lysine contains an equilibrium mixture that is more complex than previously known for any L-amino acid oxidase.