Inhibition of Tyrosine Phenol-lyase from Citrobacter freundii by 2-Azatyrosine and 3-Azatyrosine

The interactions of 2-azatyrosine and 3-azatyrosine with tyrosine phenol-lyase (TPL) from Citrobacter freundii have been examined. 2-Aza-dl-tyrosine and 3-aza-dl-tyrosine were synthesized by standard methods of amino acid synthesis, while the l-isomers were prepared from 3-hydroxypyridine and 2-hydr...

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Bibliographic Details
Published in:Biochemistry (Easton) 2001-12, Vol.40 (49), p.14862-14868
Main Authors: Watkins, E. Blake, Phillips, Robert S
Format: Article
Language:English
Subjects:
Alanine - analogs & derivatives
Alanine - chemistry
Alanine - metabolism
Alanine - pharmacology
Citrobacter freundii - enzymology
Enzyme Inhibitors - chemistry
Enzyme Inhibitors - metabolism
Enzyme Inhibitors - pharmacology
Molecular Structure
Nuclear Magnetic Resonance, Biomolecular
Tyrosine Phenol-Lyase - antagonists & inhibitors
Tyrosine Phenol-Lyase - chemistry
Tyrosine Phenol-Lyase - metabolism
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Summary:The interactions of 2-azatyrosine and 3-azatyrosine with tyrosine phenol-lyase (TPL) from Citrobacter freundii have been examined. 2-Aza-dl-tyrosine and 3-aza-dl-tyrosine were synthesized by standard methods of amino acid synthesis, while the l-isomers were prepared from 3-hydroxypyridine and 2-hydroxypyridine, respectively, with TPL (Watkins, E. B., and Phillips, R. S. (2001) Bioorg. Med. Chem. Lett. 11, 2099−2100). 3-Azatyrosine was examined as a potential transition state analogue inhibitor of TPL. Both compounds were found to be competitive inhibitors of TPL, with K i values of 3.4 mM and 135 μM for 3- and 2-aza-l-tyrosine, respectively. Thus, 3-azatyrosine does not act as a transition state analogue, possibly due to the lack of tetrahedral geometry at C-1. However, 2-aza-l-tyrosine is the most potent competitive inhibitor of TPL found to date. The K i value of 2-aza-l-tyrosine is half that of 2-aza-dl-tyrosine, indicating that the d-enantiomer is inactive as an inhibitor. Neither azatyrosine isomer was shown to be a substrate for β-elimination, based on coupled assays with lactate dehydrogenase and on HPLC measurements. Both isomers of azatyrosine form equilibrium mixtures of external aldimine and quinonoid intermediates when they bind to TPL. However, 2-azatyrosine reacts about 10-fold faster to form a quinonoid intermediate than does 3-azatyrosine. Since 2-azatyrosine is in the zwitterion or phenolate ion form at all the pH values examined, the strong binding of this compound suggests that l-tyrosine may be bound to the active site of TPL as the phenolate anion.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi015707s