Jürgen Brem

The Interaction of Nitrophenylalanines with Wild Type and Mutant 4‐Methylideneimidazole‐5‐one‐less Phenylalanine Ammonia Lyase

Racemic nitrophenylalanines and (E)‐nitrophenylacrylates are synthesized from the corresponding aldehydes. Both products are important for the examination of the mechanism of action of phenylalanine ammonia lyase (PAL). For the reaction of the rac‐nitrophenylalanines with both wild type (wt) PAL and an 4‐methylideneimidazole‐5‐one (MIO)‐less mutant, the kinetic constants Km and Vmax are determined and compared with those of the natural substrate L‐phenylalanine: the Km values for the racemic nitrophenylalanines with wt PAL are up to 9 times higher, however, the Vmax values are up to 5 times lower. Compared to wt PAL, the catalytic activity of MIO‐less PAL mutant for the deamination of L‐phenylalanine is approximately 400 times, while that for 3‐nitrophenylalanine is approximately 50 times lower. Both wt and MIO‐less PALs are enantioselective for L‐nitrophenylalanines. Thus, enantiopure D‐nitrophenylalanines can be biosynthesized from racemic substrates. The biocatalytic synthesis of the corresponding L‐enantiomers is achieved by the reverse reaction, starting from (E)‐nitrophenylacrylates. Both enantiomeric products obtained with wt and MIO‐less PAL are spectroscopically and chromatographically characterized and their optical rotations measured.