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.