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Literature summary for 4.1.99.1 extracted from

  • Phillips, R.S.; Demidkina, T.V.; Faleev, N.G.
    The role of substrate strain in the mechanism of the carbon-carbon lyases (2014), Bioorg. Chem., 57, 198-205 .
    View publication on PubMed

Crystallization (Commentary)

Crystallization (Comment) Organism
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

Protein Variants

Protein Variants Comment Organism
D133A site-directed mutagenesis, inactive mutant Proteus vulgaris
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
H458A site-directed mutagenesis, the almost inactive mutant shows 99% reduced activity compared to the wild-type enzyme Proteus vulgaris
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

Inhibitors

Inhibitors Comment Organism Structure
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
oxindolyl-L-alanine an inhibitor which is an analogue of the proposed indolenine intermediate Proteus vulgaris

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
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 ΒΌ 3:8 and D2Okcat=Km ΒΌ 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

Metals/Ions

Metals/Ions Comment Organism Structure
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
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

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-tryptophan + H2O Escherichia coli very high substrate specificity of TIL indole + pyruvate + NH3
-
?
L-tryptophan + H2O Proteus vulgaris very high substrate specificity of TIL indole + pyruvate + NH3
-
?

Organism

Organism UniProt Comment Textmining
Escherichia coli P0A853
-
-
Proteus vulgaris P28796
-
-

Reaction

Reaction Comment Organism Reaction ID
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
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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-tryptophan + H2O very high substrate specificity of TIL Escherichia coli indole + pyruvate + NH3
-
?
L-tryptophan + H2O very high substrate specificity of TIL Proteus vulgaris indole + pyruvate + NH3
-
?
additional information only the correctly protonated form of the enzyme binds the substrate, and the enzyme-substrate complex does not undergo protonation Escherichia coli ?
-
?
S-methyl-L-cysteine + H2O
-
Escherichia coli methanethiol + pyruvate + NH3
-
r

Subunits

Subunits Comment Organism
tetramer
-
Escherichia coli
tetramer
-
Proteus vulgaris

Synonyms

Synonyms Comment Organism
TIL
-
Escherichia coli
TIL
-
Proteus vulgaris
TnaA
-
Escherichia coli
TnaA
-
Proteus vulgaris
tryptophan indole lyase
-
Escherichia coli
tryptophan indole lyase
-
Proteus vulgaris

Cofactor

Cofactor Comment Organism Structure
pyridoxal 5'-phosphate dependent on, the active site residues involved in cofactor binding are highly conserved for enzyme TIL Escherichia coli
pyridoxal 5'-phosphate dependent on, the active site residues involved in cofactor binding are highly conserved for enzyme TIL Proteus vulgaris

General Information

General Information Comment Organism
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
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
additional information the conserved Phe449 in TPL locates within 3 A of the substrate aromatic ring in the closed conformation Escherichia coli
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