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Literature summary for 4.1.99.2 extracted from

  • Phillips, R.; Vita, A.; Spivey, J.; Rudloff, A.; Driscoll, M.; Hay, S.
    Ground-state destabilization by Phe-448 and Phe-449 contributes to tyrosine phenol-lyase catalysis (2016), ACS Catal., 6, 6770-6779 .
No PubMed abstract available
Search for more literature for 4.1.99.2

Cloned(Commentary)

Cloned (Comment) Organism
gene tnaA, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha Citrobacter freundii

Protein Variants

Protein Variants Comment Organism
F448A site-directed mutagenesis, the mutant shows a 104fold reduced activity with L-tyrosine compared to wild-type, stopped-flow kinetics of enzyme mutant F448A. The mutant F448A TPL forms quinonoid intermediates from L-tyrosine and S-ethyl-L-cysteine with rate constants similar to those of wild-type TPL, and can form an aminoacrylate intermediate from S-ethyl-L-cysteine but not L-tyrosine, with a rate constant similar to that of wild-type TPL Citrobacter freundii
F448H site-directed mutagenesis, enzyme mutant crystal structure with bound 3-fluoro-L-tyrosine, tense and closed conformation of F448H TPL quinonoid complex with the ligand, overview. Mutant F448H TPL has very low catalytic activity with L-tyrosine compared to wild-type Citrobacter freundii
F448L site-directed mutagenesis, the mutant shows a 103fold reduced activity with L-tyrosine compared to wild-type Citrobacter freundii
F449A site-directed mutagenesis, the mutant shows a 104fold reduced activity with L-tyrosine compared to wild-type Citrobacter freundii
Y71F site-directed mutagenesis, enzyme mutant crystal structure with bound 3-fluoro-L-tyrosine, open conformation of Y71F TPL quinonoid complex with the ligand, overview Citrobacter freundii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information pre-steady-state kinetic and steady-state kinetics, kinetic analysis of recombinant wild-type and mutant enzymes, stopped-flow kinetics of enzyme mutant F448A Citrobacter freundii

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-tyrosine + H2O Citrobacter freundii
-
 phenol + pyruvate + NH3
-
 r

Organism

Organism UniProt Comment Textmining
Citrobacter freundii P31013
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, hydrophobic interaction chromatography, and gel filtration Citrobacter freundii

Reaction

Reaction Comment Organism Reaction ID
L-tyrosine + H2O = phenol + pyruvate + NH3 mechanism of alpha,beta-elimination of L-tyrosine catalyzed by enzyme TPL, detailed overview Citrobacter freundii
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3-fluoro-L-tyrosine + H2O 3-fluoro-L-tyrosine also is a good substrate for the wild-type enzyme Citrobacter freundii 3-fluorophenol + pyruvate + NH3
-
 r
L-tyrosine + H2O
-
 Citrobacter freundii phenol + pyruvate + NH3
-
 r
L-tyrosine + H2O high activity with the wild-type enzyme Citrobacter freundii phenol + pyruvate + NH3
-
 r
additional information of the common S-alkyl-L-cysteine substrates, S-ethyl-L-cysteine has the most favorable kinetic properties with TPL Citrobacter freundii ?
-
 ?
S-(2-nitrophenyl)-L-cysteine + H2O best substrate for the wild-type enzyme Citrobacter freundii 2-nitrobenzenethiolate + pyruvate + NH3
-
 r
S-ethyl-L-cysteine + H2O
-
 Citrobacter freundii ethanethiol + pyruvate + NH3
-
 r

Subunits

Subunits Comment Organism
homotetramer 4 * 51000, recombinant enzyme, SDS-PAGE Citrobacter freundii

Synonyms

Synonyms Comment Organism
TnaA
-
 Citrobacter freundii
TPL
-
 Citrobacter freundii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Citrobacter freundii

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.000034
-
 L-tyrosine pH 8.0, 25°C, recombinant mutant F448H Citrobacter freundii
0.00016
-
 L-tyrosine pH 8.0, 25°C, recombinant mutant F449A Citrobacter freundii
0.00026
-
 L-tyrosine pH 8.0, 25°C, recombinant mutant F448A Citrobacter freundii
0.005
-
 L-tyrosine pH 8.0, 25°C, recombinant mutant F448L Citrobacter freundii
0.12
-
 S-ethyl-L-cysteine pH 8.0, 25°C, recombinant mutant F449A Citrobacter freundii
0.23
-
 S-ethyl-L-cysteine pH 8.0, 25°C, recombinant mutant F448L Citrobacter freundii
0.26
-
 S-(2-nitrophenyl)-L-cysteine pH 8.0, 25°C, recombinant mutant F448L Citrobacter freundii
0.27
-
 S-ethyl-L-cysteine pH 8.0, 25°C, recombinant mutant F448H Citrobacter freundii
0.36
-
 S-(2-nitrophenyl)-L-cysteine pH 8.0, 25°C, recombinant mutant F449A Citrobacter freundii
0.51
-
 S-ethyl-L-cysteine pH 8.0, 25°C, recombinant mutant F448A Citrobacter freundii
0.6
-
 S-(2-nitrophenyl)-L-cysteine pH 8.0, 25°C, recombinant mutant F448H Citrobacter freundii
2.5
-
 S-(2-nitrophenyl)-L-cysteine pH 8.0, 25°C, recombinant mutant F448A Citrobacter freundii
3.5
-
 L-tyrosine pH 8.0, 25°C, recombinant wild-type enzyme Citrobacter freundii
3.9
-
 S-ethyl-L-cysteine pH 8.0, 25°C, recombinant wild-type enzyme Citrobacter freundii
9.7
-
 S-(2-nitrophenyl)-L-cysteine pH 8.0, 25°C, recombinant wild-type enzyme Citrobacter freundii

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Citrobacter freundii

Cofactor

Cofactor Comment Organism Structure
pyridoxal 5'-phosphate dependent on Citrobacter freundii

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0000008
-
 L-tyrosine pH 8.0, 25°C, recombinant mutant F449A Citrobacter freundii
0.000088
-
 S-ethyl-L-cysteine pH 8.0, 25°C, recombinant mutant F449A Citrobacter freundii
0.00037
-
 L-tyrosine pH 8.0, 25°C, recombinant mutant F448H Citrobacter freundii
0.0013
-
 L-tyrosine pH 8.0, 25°C, recombinant mutant F448A Citrobacter freundii
0.0062
-
 S-(2-nitrophenyl)-L-cysteine pH 8.0, 25°C, recombinant mutant F449A Citrobacter freundii
0.035
-
 L-tyrosine pH 8.0, 25°C, recombinant mutant F448L Citrobacter freundii
0.17
-
 S-ethyl-L-cysteine pH 8.0, 25°C, recombinant mutant F448H Citrobacter freundii
0.59
-
 S-ethyl-L-cysteine pH 8.0, 25°C, recombinant wild-type enzyme Citrobacter freundii
0.965
-
 S-ethyl-L-cysteine pH 8.0, 25°C, recombinant mutant F448A Citrobacter freundii
0.994
-
 S-ethyl-L-cysteine pH 8.0, 25°C, recombinant mutant F448L Citrobacter freundii
3.3
-
 S-(2-nitrophenyl)-L-cysteine pH 8.0, 25°C, recombinant mutant F448H Citrobacter freundii
4.5
-
 S-(2-nitrophenyl)-L-cysteine pH 8.0, 25°C, recombinant mutant F448L Citrobacter freundii
9.6
-
 S-(2-nitrophenyl)-L-cysteine pH 8.0, 25°C, recombinant mutant F448A Citrobacter freundii
17.5
-
 L-tyrosine pH 8.0, 25°C, recombinant wild-type enzyme Citrobacter freundii
46
-
 S-(2-nitrophenyl)-L-cysteine pH 8.0, 25°C, recombinant wild-type enzyme Citrobacter freundii