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

Authors

  • Dr. Monica Ioana Toşa,

    1. Department of Biochemistry and Biochemical Engineering, Babeş-Bolyai University, Arany János str. 11, 400028 Cluj Napoca (Romania), Fax: (+40) 264-590818
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  • Dr. Jürgen Brem,

    1. Department of Biochemistry and Biochemical Engineering, Babeş-Bolyai University, Arany János str. 11, 400028 Cluj Napoca (Romania), Fax: (+40) 264-590818
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  • Alexandra Mantu,

    1. Department of Biochemistry and Biochemical Engineering, Babeş-Bolyai University, Arany János str. 11, 400028 Cluj Napoca (Romania), Fax: (+40) 264-590818
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  • Prof. Dr. Florin Dan Irimie,

    1. Department of Biochemistry and Biochemical Engineering, Babeş-Bolyai University, Arany János str. 11, 400028 Cluj Napoca (Romania), Fax: (+40) 264-590818
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  • Dr. Csaba Paizs,

    Corresponding author
    1. Department of Biochemistry and Biochemical Engineering, Babeş-Bolyai University, Arany János str. 11, 400028 Cluj Napoca (Romania), Fax: (+40) 264-590818
    • Department of Biochemistry and Biochemical Engineering, Babeş-Bolyai University, Arany János str. 11, 400028 Cluj Napoca (Romania), Fax: (+40) 264-590818
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  • Prof. Dr. János Rétey

    Corresponding author
    1. Institute of Organic Chemistry, Karlsruhe Institute of Technology, Richard-Willstätter-Allee, 76128 Karlsruhe (Germany), Fax: (+49) 721-608-4823
    • Institute of Organic Chemistry, Karlsruhe Institute of Technology, Richard-Willstätter-Allee, 76128 Karlsruhe (Germany), Fax: (+49) 721-608-4823
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Abstract

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.

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