Literature summary for 4.1.99.2 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 .

Search for more literature for 4.1.99.2

Crystallization (Commentary)

Crystallization (Comment)	Organism
analysis of crystal structure with bound competitive inhibitor 4-hydroxyphenylpropionate, PDB ID 1AX4	Citrobacter freundii

Protein Variants

Protein Variants	Comment	Organism
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
H343A	site-directed mutagenesis, the mutant shows altered kinetics compared to wild-type enzyme	Citrobacter freundii
R381A	site-directed mutagenesis, the mutant shows very low remaining activity compared to wild-type	Citrobacter freundii
T124A	site-directed mutagenesis, the mutant shows very low remaining activity 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

Inhibitors

Inhibitors	Comment	Organism	Structure
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]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
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

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. 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)

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

Organism

Organism	UniProt	Comment	Textmining
Citrobacter freundii	P31013	-	-

Reaction

Reaction	Comment	Organism	Reaction ID
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	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
3-fluoro-L-tyrosine + H2O	-	Citrobacter freundii	3-fluorophenol + pyruvate + NH3	-	r
L-tyrosine + H2O	very high substrate specificity of TPL	Citrobacter freundii	phenol + pyruvate + NH3	-	r

Subunits

Subunits	Comment	Organism
tetramer	-	Citrobacter freundii

Synonyms

Synonyms	Comment	Organism
beta-tyrosinase	-	Citrobacter freundii
TPL	-	Citrobacter freundii

Cofactor

Cofactor	Comment	Organism	Structure
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

General Information	Comment	Organism
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
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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Release 2023.1 (January 2023)