Phenylalanine ammonia-lyase: The use of its broad substrate specificity for mechanistic investigations and biocatalysis - Synthesis of L-arylalanines

Several fluoro- and chlorophenylalanines were found to be good substrates o f phenylalanine ammonialyase (PAL/EC 4.3.1.5) from parsley. The enantiomeri cally pure L-amino acids were obtained in goad yields by reaction of the co rresponding cinnamic acids with 5M ammonia solution (buffered to pH 10) in the presence of PAL. The kinetic constants for nine different fluoro- and c hlorophenylalanines do not provide a rigorous proof for but are consistent with the previously proposed mechanism comprising an electrophilic attack o f the methylidene-imidazolone cofactor of PAL at the aromatic nucleus as a first chemical step. In the resulting Friedel-Crafts-type sigma complex the beta-protons are activated for abstraction and consequently the pro-S is a bstracted by an enzymic base. Results from semiempirical calculations combi ned with a proposed partial active site model showed a correlation between the experimental kinetic constants and the change in polarization of the pr o-S Cd-H bond and heat of formation of the ir complexes, thus making the el ectrophilic attack at the neutral aromatic ring plausible. Furthermore, whi le 5-pyrimidinylalanine was found to be a moderately good substrate of PAL, 2-pyrimidinylalanine was an inhibitor.