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Tyrosine phenol-lyase from Citrobacter intermedius. Factors controlling substrate specificity
N G Faleev1, S B Ruvinov, T V Demidkina
1A.N. Nesmeyanov Institute of Organo-Element Compounds, Academy of Sciences of the USSR, Moscow.
European Journal of Biochemistry
|November 1, 1988
Summary
L-amino acids competitively inhibit tyrosine phenol-lyase, with inhibition correlating to side-chain hydrophobicity. Anomalously potent inhibition by aspartic and glutamic acids suggests active site interactions, while L-amino acids undergo stereospecific alpha-proton abstraction.
Area of Science:
- Biochemistry
- Enzymology
- Protein-ligand interactions
Background:
- Tyrosine phenol-lyase (TPL) is a pyridoxal phosphate-dependent enzyme.
- Understanding TPL's substrate specificity is crucial for enzyme engineering and drug development.
Purpose of the Study:
- To investigate the competitive inhibition of Citrobacter intermedius TPL by various L-amino acids.
- To elucidate the factors governing TPL substrate specificity and reaction mechanisms.
Main Methods:
- Enzyme inhibition assays using various L-amino acids and their derivatives.
- Kinetic analysis to determine inhibition constants (Ki).
- Enzymatic isotope exchange studies to probe reaction mechanisms.
Main Results:
- L-amino acids are competitive inhibitors of TPL; inhibition correlates with side-chain hydrophobicity for non-branched amino acids.
- Aspartic and glutamic acids show unusually high inhibition, suggesting specific active site interactions.
- Enzymatic isotope exchange of the alpha-proton occurs only with L-amino acids, with retention of configuration for L-phenylalanine and L-tryptophan.
- Substrate specificity is controlled by phenol elimination, requiring a para-OH group and sensitive to steric factors.
Conclusions:
- TPL active site possesses an electrophilic feature interacting with acidic amino acid side chains.
- Stereospecific alpha-proton abstraction is a key step in the reaction mechanism for L-amino acids.
- The enzyme's substrate specificity is finely tuned by the phenol elimination step and preceding proton abstraction.