Phenylalanine ammonia-lyase

 

Phenylalanine ammonia-lyase (PAL) is a plant enzyme which catalyses the non-oxidative elimination of ammonia from L-Phe to give trans-cinnamate. Trans-cinnamate is the precursor of numerous phenylpropanoid compounds and plays an important role in plant development and plant stress response. PAL may become a useful palliative for phenylketonuria as it has been shown to be able to convert excess Phe (toxic) in the blood into harmless compounds. It follows the same catalytic mechanism as tyrosine ammonia-lyase (TAL) for the deamination of L-tyrosine to (E)-4-coumarate.

 

Reference Protein and Structure

Sequence
P24481 UniProt (4.3.1.24) IPR005922, IPR022314 (Sequence Homologues) (PDB Homologues)
Biological species
Petroselinum crispum (parsley) Uniprot
PDB
1w27 - Phenylalanine ammonia-lyase (PAL) from Petroselinum crispum (1.7 Å) PDBe PDBsum 1w27
Catalytic CATH Domains
1.10.275.10 CATHdb 1.20.200.10 CATHdb (see all for 1w27)
Cofactors
2-[(1s)-1-aminoethyl]-1-carboxymethyl-5-hydroxy-4-methylimidazole (1)
Click To Show Structure

Enzyme Reaction (EC:4.3.1.24)

L-phenylalanine zwitterion
CHEBI:58095ChEBI
→
trans-cinnamate
CHEBI:15669ChEBI
+
ammonium
CHEBI:28938ChEBI
Alternative enzyme names: PAL, Phenylalanine ammonium-lyase, Phenylalanine deaminase, L-phenylalanine ammonia-lyase, Phe ammonia-lyase,

Enzyme Mechanism

Introduction

In this mechanism, the reaction proceeds via an N-MIO intermediate rather than by an FC reaction. Ammonia is eliminated from the intermediate, forming the product and regenerating the cofactor.

Catalytic Residues Roles

UniProt PDB* (1w27)
Tyr110 Tyr110A Increases acidity of the of the intermediate to promote elimination increase acidity
Tyr351 Asp351(349)A Act as a proton relay to cause the elimination of ammonia. proton relay, proton acceptor, proton donor
*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, cofactor used, overall reactant used, intermediate formation, intramolecular elimination, overall product formed, native state of cofactor regenerated, intermediate terminated, proton relay

References

  1. Weiser D et al. (2015), Chembiochem, 16, 2283-2288. Phenylalanine Ammonia-Lyase-Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles. DOI:10.1002/cbic.201500444. PMID:26345352.
  2. Pinto GP et al. (2015), Arch Biochem Biophys, 582, 107-115. New insights in the catalytic mechanism of tyrosine ammonia-lyase given by QM/MM and QM cluster models. DOI:10.1016/j.abb.2015.03.002. PMID:25772386.

Step 1. The amine group of the substrate performs a nucleophilic attack on the C=C bond of the cofactor, forming a substrate cofactor intermediate.

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Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: bimolecular nucleophilic addition, cofactor used, overall reactant used, intermediate formation

Step 2. Ty351 acts as a proton relay this leads to the elimination of ammonia, the formation of the product and the regeneration of the cofactor.

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Catalytic Residues Roles

Residue Roles
Asp351(349)A proton relay
Tyr110A increase acidity
Asp351(349)A proton acceptor, proton donor

Chemical Components

ingold: intramolecular elimination, overall product formed, native state of cofactor regenerated, intermediate terminated, proton relay
Download: Image, Marvin File

Step 1. The amine group of the substrate performs a nucleophilic attack on the C=C bond of the cofactor, forming a substrate cofactor intermediate.

Step 2. Ty351 acts as a proton relay this leads to the elimination of ammonia, the formation of the product and the regeneration of the cofactor.

Products.

Introduction

This mechanism proposal involves only a single step. There is no covalent bond between the cofactor and the substrate and the cofactor only acts to stabilize the transition state. The elimination of ammonia is facilitated by acid/base catalysis of the two tyrosine residues. This catalytic path is supported by QM/MM calculations.

Catalytic Residues Roles

UniProt PDB* (1w27)
Tyr110 Tyr110A Acts as an acid to make the ammonia a better leaving group promote heterolysis, proton donor
Tyr351 Asp351(349)A Deprotonates the substrate to facilitate the elimination of ammonia proton acceptor
*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

overall reactant used, overall product formed, bimolecular elimination, proton transfer

References

  1. Weiser D et al. (2015), Chembiochem, 16, 2283-2288. Phenylalanine Ammonia-Lyase-Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles. DOI:10.1002/cbic.201500444. PMID:26345352.
  2. Pinto GP et al. (2015), Arch Biochem Biophys, 582, 107-115. New insights in the catalytic mechanism of tyrosine ammonia-lyase given by QM/MM and QM cluster models. DOI:10.1016/j.abb.2015.03.002. PMID:25772386.

Step 1. Tyr351 deprotonates the substrate and Tyr110 protonates the amino group, this makes it a better leaving group allowing the E2 reaction to occur.

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Catalytic Residues Roles

Residue Roles
Tyr110A promote heterolysis
Tyr110A proton donor
Asp351(349)A proton acceptor

Chemical Components

overall reactant used, overall product formed, ingold: bimolecular elimination, proton transfer
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Step 1. Tyr351 deprotonates the substrate and Tyr110 protonates the amino group, this makes it a better leaving group allowing the E2 reaction to occur.

Products.

Introduction

The electrophile in the mechanism is thought to be formed by the autocatalytic formation of 3,5-dihydro-5-methyldiene-4H-imidazole-4-one group (MIO) from the tripeptide Ala202-Ser203-Gly204. The MIO group is formed by cyclisation as a result of two water elimination steps. The sp3 conformation at Gly204 amide N increases the electrophicity of the Ser203 beta-C atom. In the reaction mechanism, an electron pair of the substrate phenyl ring attacks the beta-C of Ser203 of the MIO causing the MIO to become aromatic. The conversion of MIO to the aromatic state changes the conformation of Gly204 amide-N from sp3 to sp2, causing a small peptide displacement. The Ser203 oxygen anion produced stabilises the positively charged sigma-complex of the substrate phenyl group. The positive charge is also stabilised by an interaction with the pi-electrons of Phe400. The electron-deficient phenyl ring of L-Phe renders the two hydrogen atoms at the beta-C atom of the substrate acidic. Tyr351' from another subunit abstracts the pro-S beta-hydrogen from the substrate. The rearrangement of the Phe-MIO adduct eliminates the amino group and regenerates MIO. The resulting ammonia is released into the solvent after the trans-cinnamate has diffused away. The proton abstracted by Tyr351' can be easily released to the bulk solvent to regenerate the enzyme.

Catalytic Residues Roles

UniProt PDB* (1w27)
Tyr351 Asp351(349)A Tyr351' acts as a base, abstracting a beta-H from the substrate.The proton is released into the bulk solvent. proton acceptor, proton donor
Phe400 Gly400(398)A Phe400 stabilises the positively charged sigma-complex intermediate to prevent the removal of the proton in the ortho-position of the aromatic ring. 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

aromatic bimolecular electrophilic addition, intermediate formation, overall reactant used, cofactor used, proton transfer, intramolecular elimination, overall product formed, native state of cofactor regenerated, native state of enzyme regenerated, intermediate terminated

References

  1. Ritter H et al. (2004), Plant Cell, 16, 3426-3436. Structural Basis for the Entrance into the Phenylpropanoid Metabolism Catalyzed by Phenylalanine Ammonia-Lyase. DOI:10.1105/tpc.104.025288. PMID:15548745.
  2. Weiser D et al. (2015), Chembiochem, 16, 2283-2288. Phenylalanine Ammonia-Lyase-Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles. DOI:10.1002/cbic.201500444. PMID:26345352.
  3. Röther D et al. (2002), Eur J Biochem, 269, 3065-3075. An active site homology model of phenylalanine ammonia-lyase fromP. crispum. DOI:10.1046/j.1432-1033.2002.02984.x. PMID:12071972.

Step 1. The phenyl ring attacks the C=C bond of the MIO cofactor forming a substrate cofactor intermediate. The resulting carbocation is stabilized by Phe400.

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Catalytic Residues Roles

Residue Roles
Gly400(398)A electrostatic stabiliser

Chemical Components

ingold: aromatic bimolecular electrophilic addition, intermediate formation, overall reactant used, cofactor used

Step 2. Tyr351 deprotonates the beta carbon of the substrate, neutralizing the carbocation.

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Catalytic Residues Roles

Residue Roles
Gly400(398)A electrostatic stabiliser
Asp351(349)A proton acceptor

Chemical Components

proton transfer

Step 3. Ammonia is eliminated from the substrate and the cofactor is regenerated.

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Catalytic Residues Roles

Residue Roles
Asp351(349)A proton donor

Chemical Components

ingold: intramolecular elimination, overall product formed, native state of cofactor regenerated, native state of enzyme regenerated, proton transfer, intermediate terminated
Download: Image, Marvin File

Step 1. The phenyl ring attacks the C=C bond of the MIO cofactor forming a substrate cofactor intermediate. The resulting carbocation is stabilized by Phe400.

Step 2. Tyr351 deprotonates the beta carbon of the substrate, neutralizing the carbocation.

Step 3. Ammonia is eliminated from the substrate and the cofactor is regenerated.

Products.

Contributors

Gemma L. Holliday, James Willey, Noa Marson, Yordanos Abeje