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Phenylalanine Ammonia‐Lyase: The Use of Its Broad Substrate Specificity for Mechanistic Investigations and Biocatalysis—Synthesis of L ‐Arylalanines
Author(s) -
Gloge Andreas,
Zoń Jerzy,
Kövári Ágnes,
Poppe László,
Rétey János
Publication year - 2000
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/1521-3765(20000915)6:18<3386::aid-chem3386>3.0.co;2-5
Subject(s) - biocatalysis , lyase , substrate (aquarium) , substrate specificity , chemistry , ammonia , stereochemistry , biochemistry , enzyme , biology , catalysis , reaction mechanism , ecology
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 C β −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.