Phenylalanine Ammonia-Lyase: The Use of Its Broad Substrate Specificity for Mechanistic Investigations and Biocatalysis—Synthesis ofL-Arylalanines

Several fluoro- and chloro-phenylalanines were found to be good substrates of phenylalanine ammonia-lyase (PAL/EC 4.3.1.5) from parsley. The enantiomerically pure L-amino acids were obtained in good yields by reaction of the corresponding cinnamic acids with 5 M ammonia solution (buffered to pH 10) in the presence of PAL. The kinetic constants for nine different fluoro- and chlorophenylalanines do not provide a rigorous proof for but are consistent with the previously proposed mechanism comprising an electrophilic attack of the methylidene-imidazolone cofactor of PAL at the aromatic nucleus as a first chemical step. In the resulting Friedel–Crafts-type σ complex the β-protons are activated for abstraction and consequently the pro-S is abstracted by an enzymic base. Results from semiempirical calculations combined with a proposed partial active site model showed a correlation between the experimental kinetic constants and the change in polarization of the pro-S Cβ−H bond and heat of formation of the σ complexes, thus making the electrophilic attack at the neutral aromatic ring plausible. Furthermore, while 5-pyrimidinylalanine was found to be a moderately good substrate of PAL, 2-pyrimidinylalanine was an inhibitor.