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Literature summary extracted from
- The role of substrate strain in the mechanism of the carbon-carbon lyases (2014), Bioorg. Chem., 57, 198-205 .
Crystallization (Commentary)
| EC Number | Crystallization (Comment) | Organism |
|---|---|---|
| 4.1.99.1 | analysis of the enzyme tetramer with one pyridoxal 5'-phosphate bound to each monomer, crystal structure at 2.1 A resolution, PDB ID 1AX4 | Proteus vulgaris |
| 4.1.99.2 | analysis of crystal structure with bound competitive inhibitor 4-hydroxyphenylpropionate, PDB ID 1AX4 | Citrobacter freundii |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 4.1.99.1 | D133A | site-directed mutagenesis, inactive mutant | Proteus vulgaris |
| 4.1.99.1 | F448H | site-directed mutagenesis, the imidazole of F448H TPL forms a hydrogen bond to the substrate, consistent with the histidine being capable of hydrogen bonding to the substrate in TIL | Proteus vulgaris |
| 4.1.99.1 | H458A | site-directed mutagenesis, the almost inactive mutant shows 99% reduced activity compared to the wild-type enzyme | Proteus vulgaris |
| 4.1.99.1 | H463F | site-directed mutagenesis, the mutation results in a 103fold decrease in tryptophan elimination activity and in a 1000fold decrease in tryptophan elimination activity and loss of the pKa of 6.0 in the pH dependence of kcat/Km, suggesting that His463 is that base. In contrast, kcat is pH-independent, demonstrating that only the correctly protonated form of the enzyme binds the substrate, and the enzyme-substrate complex does not undergo protonation | Escherichia coli |
| 4.1.99.2 | 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 | Citrobacter freundii |
| 4.1.99.2 | H343A | site-directed mutagenesis, the mutant shows altered kinetics compared to wild-type enzyme | Citrobacter freundii |
| 4.1.99.2 | R381A | site-directed mutagenesis, the mutant shows very low remaining activity compared to wild-type | Citrobacter freundii |
| 4.1.99.2 | T124A | site-directed mutagenesis, the mutant shows very low remaining activity compared to wild-type | Citrobacter freundii |
| 4.1.99.2 | 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 |
Inhibitors
| EC Number | Inhibitors | Comment | Organism | Structure |
|---|---|---|---|---|
| 4.1.99.1 | oxindolyl-L-alanine | an inhibitor which is an analogue of the proposed indolenine intermediate. The pH dependence of Ki for oxindolyl-L-alanine exhibits two basic pKas of 6.0 and 7.6 | Escherichia coli |  |
| 4.1.99.1 | oxindolyl-L-alanine | an inhibitor which is an analogue of the proposed indolenine intermediate | Proteus vulgaris | |
| 4.1.99.2 | 4-hydroxyphenylpropionate | competitive inhibitor, addition of 4-hydroxypyridin in the reaction results in decay of the quinonoid-aldimine spectrum with rate constant of 1.4/s and formation of an intermediate absorbing at 338 nm, assigned to an aminoacrylate intermediate | Citrobacter freundii | |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 4.1.99.1 | additional information | - |
additional information | pre-steady state kinetics and steady state kinetic study, stopped-flow measurements and stopped-flow spectra of the reaction of TIL with L-tryptophan, overview. The pH dependence of kcat/Km of Escherichia coli TIL for tryptophan exhibits 2 basic groups, with pKas of 6.0 and 7.6. The base with pKa of 7.6 is involved in the deprotonation of the alpha-carbon of substrates, and the base with pKa of 6.0 activates the indole ring of the tryptophan substrate for elimination. There is a pH-independent primary isotope effect on kcat (Dkcat = 2.5) and kcat/Km (Dkcat/Km = 2.8) for alpha-[2H]-L-tryptophan, indicating that a step (or steps) involving transfer of the alpha-proton is partially rate-limiting. The TIL reaction shows pD-independent solvent isotope effects in D2O (D2Okcat ¼ 3:8 and D2Okcat=Km ¼ 2:8), and the substrate isotope effect is reduced in D2O (Dkcat = 1.25 and Dkcat/Km = 1.82), suggesting that the steady-state solvent and substrate isotope effects are on different steps. The proton inventory for the reaction of TIL is concave downward, indicating that multiple waters are involved in the transition state of the solvent sensitive step | Escherichia coli | |
| 4.1.99.2 | additional information | - |
additional information | pre-steady state kinetics and steady state kinetic study, overview. TPL from Citrobacter freundii exhibits two basic pKas, with an average value of 7.8, in the pH dependence of kcat/Km, and kcat is pH-independent. The TPL reaction shows primary isotope effects of about 3 on kcat and 2 on kcat/Km for both enzymes, also suggesting that steps involving transfer of the alpha-proton are partially rate-limiting. The primary isotope effect increases to 5.4 and 3.8, respectively, for kcat and kcat/Km with alpha-[2H]-3-fluoro-L-tyrosine with H343A TPL, compared to 3.9 and 2.2 for wild-type TPL. Stopped-flow measurements, the reaction of TPL with L-tyrosine and 3-fluoro-L-tyrosine in the stopped-flow spectrophotometer also shows rapid formation of quinonoid intermediate | Citrobacter freundii |
Metals/Ions
| EC Number | Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|---|
| 4.1.99.1 | additional information | the enzyme requires a monovalent cation, either K+, NH4+, Rb+ or Cs+ for activity, with Na+ and Li+ giving little or no activity | Escherichia coli | |
| 4.1.99.1 | additional information | the enzyme requires a monovalent cation, either K+, NH4+, Rb+ or Cs+ for activity, with Na+ and Li+ giving little or no activity | Proteus vulgaris | |
| 4.1.99.2 | additional information | the enzyme requires a monovalent cation, either K+, NH4+, Rb+ or Cs+ for activity, with Na+ and Li+ giving little or no activity. The cation is bound by the gamma-carboxylate of a conserved glutamate residue Glu69 of the TPL peptide backbone carbonyl oxygens, and waters, and is located about 10 A from cofactor pyridoxal 5'-phosphate | Citrobacter freundii |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 4.1.99.1 | L-tryptophan + H2O | Escherichia coli | very high substrate specificity of TIL | indole + pyruvate + NH3 | - |
? | |
| 4.1.99.1 | L-tryptophan + H2O | Proteus vulgaris | very high substrate specificity of TIL | indole + pyruvate + NH3 | - |
? | |
| 4.1.99.2 | L-tyrosine + H2O | Citrobacter freundii | very high substrate specificity of TPL | phenol + pyruvate + NH3 | - |
r | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 4.1.99.1 | Escherichia coli | P0A853 | - |
- |
| 4.1.99.1 | Proteus vulgaris | P28796 | - |
- |
| 4.1.99.2 | Citrobacter freundii | P31013 | - |
- |
Reaction
| EC Number | Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|---|
| 4.1.99.1 | L-tryptophan + H2O = indole + pyruvate + NH3 | an active site base is essential for activity, and alpha-deuterated substrate exhibits modest primary isotope effects on kcat and kcat/Km, suggesting that substrate deprotonation is partially rate-limiting. Pre-steady state kinetics with enzyme TIL show rapid formation of an external aldimine intermediate, followed by deprotonation to give a quinonoid intermediate absorbing at about 500 nm. The mechanism of TIL requires both substrate strain and acid/base catalysis, and substrate strain is probably responsible for the very high substrate specificity of TIL. Acid-base catalysis mechanism of TIL, overview. The indole ring is preorganized into the active conformation before alpha-deprotonation occurs | Escherichia coli | |
| 4.1.99.1 | L-tryptophan + H2O = indole + pyruvate + NH3 | an active site base is essential for activity, and alpha-deuterated substrate exhibits modest primary isotope effects on kcat and kcat/Km, suggesting that substrate deprotonation is partially rate-limiting. Pre-steady state kinetics with enzyme TIL show rapid formation of external an aldimine intermediate, followed by deprotonation to give a quinonoid intermediate absorbing at about 500 nm. The mechanism of TIL requires both substrate strain and acid/base catalysis, and substrate strain is probably responsible for the very high substrate specificity of TIL. Acid-base catalysis mechanism of TIL, overview. The indole ring is preorganized into the active conformation before alpha-deprotonation occurs | Proteus vulgaris | |
| 4.1.99.2 | L-tyrosine + H2O = phenol + pyruvate + NH3 | an active site base is essential for activity, and alpha-deuterated substrate exhibits modest primary isotope effects on kcat and kcat/Km, suggesting that substrate deprotonation is partially rate-limiting. Pre-steady state kinetics with enzyme TPL show rapid formation of external aldimine intermediate, followed by deprotonation to give a quinonoid intermediate absorbing at about 500 nm. The mechanism of TPL requires both substrate strain and acid/base catalysis, and substrate strain is probably responsible for the very high substrate specificity of TPL. Acid-base catalysis mechanism of TPL, overview | Citrobacter freundii |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 4.1.99.1 | L-tryptophan + H2O | very high substrate specificity of TIL | Escherichia coli | indole + pyruvate + NH3 | - |
? | |
| 4.1.99.1 | L-tryptophan + H2O | very high substrate specificity of TIL | Proteus vulgaris | indole + pyruvate + NH3 | - |
? | |
| 4.1.99.1 | additional information | only the correctly protonated form of the enzyme binds the substrate, and the enzyme-substrate complex does not undergo protonation | Escherichia coli | ? | - |
? | |
| 4.1.99.1 | S-methyl-L-cysteine + H2O | - |
Escherichia coli | methanethiol + pyruvate + NH3 | - |
r | |
| 4.1.99.2 | 3-fluoro-L-tyrosine + H2O | - |
Citrobacter freundii | 3-fluorophenol + pyruvate + NH3 | - |
r | |
| 4.1.99.2 | L-tyrosine + H2O | very high substrate specificity of TPL | Citrobacter freundii | phenol + pyruvate + NH3 | - |
r | |
Subunits
| EC Number | Subunits | Comment | Organism |
|---|---|---|---|
| 4.1.99.1 | tetramer | - |
Escherichia coli |
| 4.1.99.1 | tetramer | - |
Proteus vulgaris |
| 4.1.99.2 | tetramer | - |
Citrobacter freundii |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 4.1.99.1 | TIL | - |
Escherichia coli |
| 4.1.99.1 | TIL | - |
Proteus vulgaris |
| 4.1.99.1 | TnaA | - |
Escherichia coli |
| 4.1.99.1 | TnaA | - |
Proteus vulgaris |
| 4.1.99.1 | tryptophan indole lyase | - |
Escherichia coli |
| 4.1.99.1 | tryptophan indole lyase | - |
Proteus vulgaris |
| 4.1.99.2 | beta-tyrosinase | - |
Citrobacter freundii |
| 4.1.99.2 | TPL | - |
Citrobacter freundii |
Cofactor
| EC Number | Cofactor | Comment | Organism | Structure |
|---|---|---|---|---|
| 4.1.99.1 | pyridoxal 5'-phosphate | dependent on, the active site residues involved in cofactor binding are highly conserved for enzyme TIL | Escherichia coli | |
| 4.1.99.1 | pyridoxal 5'-phosphate | dependent on, the active site residues involved in cofactor binding are highly conserved for enzyme TIL | Proteus vulgaris | |
| 4.1.99.2 | pyridoxal 5'-phosphate | dependent on, the active site residues involved in cofactor binding are highly conserved for enzyme TPL. Pyridoxal 5'-phosphate is bound to the internal aldimine by a lysine epsilon-amino group of Lys257 and the protonated pyridine ring forms an ionic/hydrogen bond with an aspartate beta-carboxylate of Asp214 | Citrobacter freundii | |
General Information
| EC Number | General Information | Comment | Organism |
|---|---|---|---|
| 4.1.99.1 | evolution | the carbon-carbon lyases, tryptophan indole lyase (TIL) and tyrosine phenol-lyase (TPL, EC 4.1.99.2) are bacterial enzymes which catalyze the reversible elimination of indole and phenol from L-tryptophan and L-tyrosine, respectively. These pyridoxal 5'-phosphate-dependent enzymes show high sequence homology (about 40% identity) and both form homotetrameric structures. Pre-steady state kinetics with TPL and TIL show rapid formation of external aldimine intermediates, followed by deprotonation to give quinonoid intermediates absorbing at about 500 nm. The active sites of TIL and TPL are highly conserved with the exceptions of these residues: Arg381(TPL)/Ile396 (TIL), Thr124 (TPL)/Asp137 (TIL), and Phe448 (TPL)/His463 (TIL). The conserved tyrosine, Tyr71 (TPL)/Tyr74 (TIL) is essential for elimination activity with both enzymes, and likely plays a role as a proton donor to the leaving group. A unique feature of TIL and TPL is another strictly conserved lysine immediately preceding the pyridoxal 5'-phosphate-binding lysine, and hydrogen bonded to a water molecule bound to the monovalent cation. The mechanisms of TPL and TIL require both substrate strain and acid/base catalysis, and substrate strain is probably responsible for the very high substrate specificity of TPL and TIL. Both enzymes require a monovalent cation, either K+, NH4+, Rb+ or Cs+ for activity, with Na+ and Li+ giving little or no activity. The active site residues involved in PLP binding are highly conserved for both TIL and TPL. Sequence comparisons | Escherichia coli |
| 4.1.99.1 | evolution | the carbon-carbon lyases, tryptophan indole lyase (TIL) and tyrosine phenol-lyase (TPL, EC 4.1.99.2) are bacterial enzymes which catalyze the reversible elimination of indole and phenol from L-tryptophan and L-tyrosine, respectively. These pyridoxal 5'-phosphate-dependent enzymes show high sequence homology (about 40% identity) and both form homotetrameric structures. Pre-steady state kinetics with TPL and TIL show rapid formation of external aldimine intermediates, followed by deprotonation to give quinonoid intermediates absorbing at about 500 nm. The active sites of TIL and TPL are highly conserved with the exceptions of these residues: Arg381(TPL)/Ile396 (TIL), Thr124 (TPL)/Asp137 (TIL), and Phe448 (TPL)/His463 (TIL). The conserved tyrosine, Tyr71 (TPL)/Tyr74 (TIL) is essential for elimination activity with both enzymes, and likely plays a role as a proton donor to the leaving group. A unique feature of TIL and TPL is another strictly conserved lysine immediately preceding the pyridoxal 5'-phosphate-binding lysine, and hydrogen bonded to a water molecule bound to the monovalent cation. The mechanisms of TPL and TIL require both substrate strain and acid/base catalysis, and substrate strain is probably responsible for the very high substrate specificity of TPL and TIL. Both enzymes require a monovalent cation, either K+, NH4+, Rb+ or Cs+ for activity, with Na+ and Li+ giving little or no activity. The active site residues involved in PLP binding are highly conserved for both TIL and TPL. Sequence comparisons | Proteus vulgaris |
| 4.1.99.1 | additional information | the conserved Phe449 in TPL locates within 3 A of the substrate aromatic ring in the closed conformation | Escherichia coli |
| 4.1.99.1 | additional information | the conserved Phe459 in TPL locates within 3 A of the substrate aromatic ring in the closed conformation. Asp133 may be the residue that contacts the substrate | Proteus vulgaris |
| 4.1.99.2 | evolution | the carbon-carbon lyases, tryptophan indole lyase (TIL, EC 4.1.99.1) and tyrosine phenol-lyase (TPL) are bacterial enzymes which catalyze the reversible elimination of indole and phenol from L-tryptophan and L-tyrosine, respectively. These pyridoxal 5'-phosphate-dependent enzymes show high sequence homology (about 40% identity) and both form homotetrameric structures. Pre-steady state kinetics with TPL and TIL show rapid formation of external aldimine intermediates, followed by deprotonation to give quinonoid intermediates absorbing at about 500 nm. The active sites of TIL and TPL are highly conserved with the exceptions of these residues: Arg381(TPL)/Ile396 (TIL), Thr124 (TPL)/Asp137 (TIL), and Phe448 (TPL)/His463 (TIL). The conserved tyrosine, Tyr71 (TPL)/Tyr74 (TIL) is essential for elimination activity with both enzymes, and likely plays a role as a proton donor to the leaving group.A unique feature of TIL and TPL is another strictly conserved lysine (Lys256 in Citrobacter freundii TPL) immediately preceding the pyridoxal 5'-phosphate-binding lysine, and hydrogen bonded to a water molecule bound to the monovalent cation. The mechanisms of TPL and TIL require both substrate strain and acid/base catalysis, and substrate strain is probably responsible for the very high substrate specificity of TPL and TIL. Both enzymes require a monovalent cation, either K+, NH4+, Rb+ or Cs+ for activity, with Na+ and Li+ giving little or no activity. The active site residues involved in PLP binding are highly conserved for both TIL and TPL. Sequence comparisons | Citrobacter freundii |
| 4.1.99.2 | additional information | essential catalytic residues are Tyr71, Thr124, Arg381, and Phe448. There are two quinonoid structures of the enzyme, relaxed and tense. In the relaxed structure, the substrate aromatic ring is in plane with the Cbeta-Cgamma bond, but in the tense structure, the substrate aromatic ring is about 20° out of plane with the Cbeta-Cgamma bond. In the tense structure, hydrogen bonds are formed between the substrate OH and the guanidinium of Arg381 and the OH of Thr124, and the phenyl rings of Phe448 and 449 provide steric strain. Structure of the active site of the TPL quinonoid complex with L-alanine and pyridine N-oxide, overview | Citrobacter freundii |
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