M-CSA Mechanism and Catalytic Site Atlas

Tyrosine phenol-lyase

Tyrosine phenol-lyases (EC:4.1.99.2) (beta-tyrosinase), are pyridoxal-phosphate enzymes belonging to the beta-eliminating lyase family. They catalyse the reversible cleavage of the Cβ–Cγ bond of L-Tyr to produce phenol, ammonium, and pyruvate.

Reference Protein and Structure

Sequence: P31013 (4.1.99.2) (Sequence Homologues) (PDB Homologues)
Biological species: Citrobacter freundii (Bacteria)
PDB: 2tpl - TYROSINE PHENOL-LYASE FROM CITROBACTER INTERMEDIUS COMPLEX WITH 3-(4'-HYDROXYPHENYL)PROPIONIC ACID, PYRIDOXAL-5'-PHOSPHATE AND CS+ ION (2.5 Å)
Catalytic CATH Domains: 3.40.640.10 3.90.1150.10 (see all for 2tpl)
Cofactors: Pyridoxal 5'-phosphate(2-) (1)

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Enzyme Reaction (EC:4.1.99.2)

water

CHEBI:15377

L-tyrosine zwitterion

CHEBI:58315

→

pyruvate

CHEBI:15361

phenol

CHEBI:15882

ammonium

CHEBI:28938

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: Beta-tyrosinase, L-tyrosine phenol-lyase (deaminating),

Enzyme Mechanism

Mechanism Proposal 1 ☆☆☆

Summary
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
Step 7
Step 8
Step 9
Step 10
Step 11
Products
All Steps

Introduction

The tyrosine substrate initiates the first transaldimination reaction to displace the Lys257. Lysine then abstracts a proton from the substrate with PLP acting as an electron sink. A second proton transfer occurs (with Tyr71B) which activates the phenol moiety for elimination, which occurs next. The resulting 2-aminoprop-2-enoate product is released by a second transaldimination. 2-aminoprop-2-enoate then undergoes tautomerisation and subsequent hydrolysis, most likely outside the active site.

Catalytic Residues Roles

UniProt	PDB* (2tpl)
Phe448	Phe448A	Introduces steric strain in the substrates, destabilising the ground state and stabilising the quinoid intermediates.	ground state destabiliser, electrostatic stabiliser
Tyr71	Tyr71B	Acts as a general acid/base.	proton acceptor, proton donor
Lys257	Llp257A	This residue is covalently attached to the PLP cofactor in the ground state of the enzyme. It acts as a general acid/base during the course of the reaction, and is responsible for the transaldimination that releases the final product from the cofactor to regenerate the active site.	covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor, electron pair acceptor, electron pair donor
Phe123	Phe123A	Ensures that when the PLP cofactor is no longer covalently attached to the enzyme it remains in the correct orientation for the reaction to occur.	steric role
Asp214	Asp214A	Forms a hydrogen bond with the nitrogen of the pyrimidine ring of the PLP cofactor, stabilising the cofactor and allowing it to act as an electron sink.	electrostatic stabiliser
Thr124, Arg381	Thr124A, Arg381A	Helps stabilise the reactive intermediates and transition states formed during the course of the reaction.	electrostatic stabiliser

*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular nucleophilic addition, proton transfer, overall reactant used, schiff base formed, unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, overall product formed, enzyme-substrate complex formation, native state of cofactor regenerated, native state of enzyme regenerated, tautomerisation (not keto-enol), reaction occurs outside the enzyme, intramolecular elimination

References

Milić D et al. (2011), J Am Chem Soc, 133, 16468-16476. Crystallographic Snapshots of Tyrosine Phenol-lyase Show That Substrate Strain Plays a Role in C–C Bond Cleavage. DOI:10.1021/ja203361g. PMID:21899319.
Milić D et al. (2008), J Biol Chem, 283, 29206-29214. Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates. DOI:10.1074/jbc.M802061200. PMID:18715865.
Milić D et al. (2006), Biochemistry, 45, 7544-7552. Structures of apo- and holo-tyrosine phenol-lyase reveal a catalytically critical closed conformation and suggest a mechanism for activation by K+ ions. DOI:10.1021/bi0601858. PMID:16768450.
Barbolina MV et al. (2000), Protein Eng, 13, 207-215. Citrobacter freundii tyrosine phenol-lyase: the role of asparagine 185 in modulating enzyme function through stabilization of a quinonoid intermediate. DOI:10.1093/protein/13.3.207. PMID:10775663.
Sundararaju B et al. (1997), Biochemistry, 36, 6502-6510. The crystal structure of Citrobacter freundii tyrosine phenol-lyase complexed with 3-(4'-hydroxyphenyl)propionic acid, together with site-directed mutagenesis and kinetic analysis, demonstrates that arginine 381 is required for substrate specificity. DOI:10.1021/bi962917+. PMID:9174368.

Step 1. In the first step of the initial transaldimination reaction the substrate tyrosine initiates a nucleophilic attack on the PLP cofactor.

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Catalytic Residues Roles

Residue	Roles
Phe123A	steric role
Thr124A	electrostatic stabiliser
Asp214A	electrostatic stabiliser
Arg381A	electrostatic stabiliser
Phe448A	ground state destabiliser
Llp257A	covalently attached, proton acceptor, electron pair acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, overall reactant used

Step 2. In the final step of the transaldimination Lys257 is eliminated from the PLP cofactor.

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Catalytic Residues Roles

Residue	Roles
Thr124A	electrostatic stabiliser
Asp214A	electrostatic stabiliser
Arg381A	electrostatic stabiliser
Phe448A	electrostatic stabiliser
Phe123A	steric role
Llp257A	nucleofuge

Chemical Components

schiff base formed, ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage

Step 3. Lys257 abstracts a proton from the covalently bound intermediate, which initiates a double bond rearrangement in which PLP acts as an electron sink.

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Catalytic Residues Roles

Residue	Roles
Thr124A	electrostatic stabiliser
Asp214A	electrostatic stabiliser
Arg381A	electrostatic stabiliser
Phe448A	electrostatic stabiliser
Phe123A	steric role
Llp257A	proton acceptor

Chemical Components

proton transfer

Step 4. A highly unstable quinoid species is formed by deprotonation of Tyr71B.

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Catalytic Residues Roles

Residue	Roles
Thr124A	electrostatic stabiliser
Asp214A	electrostatic stabiliser
Arg381A	electrostatic stabiliser
Phe448A	electrostatic stabiliser
Phe123A	steric role
Tyr71B	proton donor

Chemical Components

proton transfer

Step 5. The PLP cofactor initiates a double bond rearrangement that results in the elimination of the phenol group.

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Catalytic Residues Roles

Residue	Roles
Thr124A	electrostatic stabiliser
Asp214A	electrostatic stabiliser
Arg381A	electrostatic stabiliser
Phe448A	electrostatic stabiliser
Phe123A	steric role

Chemical Components

enzyme-substrate complex cleavage, ingold: unimolecular elimination by the conjugate base, overall product formed

Step 6. Tyr71B abstracts a proton from Lys257A, activating it for the reverse transaldimination reaction.

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Catalytic Residues Roles

Residue	Roles
Phe123A	steric role
Asp214A	electrostatic stabiliser
Tyr71B	proton acceptor
Llp257A	proton donor

Chemical Components

proton transfer

Step 7. Lys257A initiates a nucleophilic attack on the PLP cofactor in the first step of the final transaldimination reaction.

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Catalytic Residues Roles

Residue	Roles
Phe123A	steric role
Asp214A	electrostatic stabiliser
Llp257A	proton donor, nucleophile

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation

Step 8. Lys257A eliminates the 2-aminoprop-2-enoate enzyme product.

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Catalytic Residues Roles

Residue	Roles
Asp214A	electrostatic stabiliser
Phe123A	steric role
Llp257A	electron pair donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, native state of cofactor regenerated, native state of enzyme regenerated, schiff base formed, enzyme-substrate complex cleavage

Step 9. It is likely that the rest of the mechanism occurs outside of the active site. The product undergoes tautomerisation.

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Catalytic Residues Roles

Residue	Roles

Chemical Components

tautomerisation (not keto-enol), reaction occurs outside the enzyme

Step 10. Water initiates a nucleophilic attack on the imine carbon.

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Catalytic Residues Roles

Residue	Roles

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, reaction occurs outside the enzyme

Step 11. The final products are formed by an intramolecular elimination.

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Catalytic Residues Roles

Residue	Roles

Chemical Components

ingold: intramolecular elimination, proton transfer, reaction occurs outside the enzyme, overall product formed