L-serine ammonia-lyase

 

serine racemase (SRR) catalyses the PLP-dependent synthesis of D-serine from L-serine (and vice versa. SRR also has dehydratase activity towards both L-serine and D-serine, resulting in pyruvate and ammonia. It is allosterically activated by ATP, by magnesium, and possibly also by other divalent metal cations. The magnesium ion present in the crystal structure is thought to be essential for the structural integrity of the enzymes, and not directly involved in catalysis [PMID:20564571].

 

Reference Protein and Structure

Sequence
O59791 UniProt (4.3.1.17, 4.3.1.18, 5.1.1.18) IPR001926 (Sequence Homologues) (PDB Homologues)
Biological species
Schizosaccharomyces pombe 972h- (Fission yeast) Uniprot
PDB
2zr8 - Crystal Structure of Modified Serine Racemase complexed with Serine (2.2 Å) PDBe PDBsum 2zr8
Catalytic CATH Domains
3.40.50.1100 CATHdb (see all for 2zr8)
Cofactors
Magnesium(2+) (1), Pyridoxal 5'-phosphate(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:4.3.1.17)

L-serine zwitterion
CHEBI:33384ChEBI
→
ammonium
CHEBI:28938ChEBI
+
pyruvate
CHEBI:15361ChEBI
Alternative enzyme names: L-hydroxyaminoacid dehydratase, L-serine deaminase, L-serine dehydratase, L-serine hydro-lyase (deaminating), Serine deaminase,

Enzyme Mechanism

Introduction

The neutral amine group of L-serine attacks the imine functionality of the pyridoxal-5-phosphate cofactor, forming a Schiff base precursor. The tetrahedral intermediate collapses, generating the external aldimine, PDD-substrate complex. Lys57 acts as a general base towards the C-alpha of the covalently bound serine, forming a planar sterocentre [PMID:19640845]. Beta-elimination of water from the carbaion can occur to form an amino-acrylate intermediate. This undergoes non-enzymatic hydrolysis to form water, ammonia and a lysino-alanine modified PLP cofactor which is still capable of isomerase activity [PMID:19155267, PMID:19640845]. Lys57 then acts as a nucleophile to regenerate the cofactor and the final enzymatic product. It is thought that the final hydrolysis occurs outside of the enzyme active site.

Catalytic Residues Roles

UniProt PDB* (2zr8)
Ser82 Ser82A Suggested to act as a general acid/base during the course of the reaction. electrostatic stabiliser
Lys57 Lys57A Lys57 is covalently attached to the PLP cofactor in the ground state of the reaction. It is thought to act as a general acid/base during the course of the reaction. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor, electron pair acceptor, electron pair donor
Ser308 Ser308A Acts to stabilise the reactive intermediates and transition states during the course of the reaction. electrostatic stabiliser
Gly212 (main-C), Asp214, Glu208 Gly212A (main-C), Asp214A, Glu208A Forms part of the magnesium binding site. metal ligand
*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

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

References

  1. Goto M et al. (2009), J Biol Chem, 284, 25944-25952. Crystal Structure of a Homolog of Mammalian Serine Racemase from Schizosaccharomyces pombe. DOI:10.1074/jbc.m109.010470. PMID:19640845.
  2. Nitoker N et al. (2015), Biochemistry, 54, 516-527. Understanding the reaction mechanism and intermediate stabilization in mammalian serine racemase using multiscale quantum-classical simulations. DOI:10.1021/bi500984m. PMID:25493718.
  3. Yamauchi T et al. (2009), J Biochem, 145, 421-424. Serine Racemase with Catalytically Active Lysinoalanyl Residue*. DOI:10.1093/jb/mvp010. PMID:19155267.

Step 1. The neutral amine group of L-serine attacks the imine functionality of the pyridoxal-5-phosphate cofactor, forming a Schiff base precursor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu208A metal ligand
Gly212A (main-C) metal ligand
Asp214A metal ligand
Lys57A covalently attached
Ser82A electrostatic stabiliser
Ser308A electrostatic stabiliser
Lys57A proton acceptor, electron pair acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used, schiff base formed

Step 2. The tetrahedral intermediate collapses, generating the external aldimine, PDD-substrate complex.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu208A metal ligand
Gly212A (main-C) metal ligand
Asp214A metal ligand
Lys57A covalently attached
Ser82A electrostatic stabiliser
Ser308A electrostatic stabiliser
Lys57A nucleofuge

Chemical Components

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

Step 3. Lys57 acts as a general base towards the C-alpha of the covalently bound serine, forming a planar sterocentre [PMID:19640845].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu208A metal ligand
Gly212A (main-C) metal ligand
Asp214A metal ligand
Ser82A electrostatic stabiliser
Ser308A electrostatic stabiliser
Lys57A proton acceptor

Chemical Components

proton transfer

Step 4. Beta-elimination of water from the carbaion can occur to form an amino-acrylate intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu208A metal ligand
Gly212A (main-C) metal ligand
Asp214A metal ligand
Ser82A electrostatic stabiliser
Ser308A electrostatic stabiliser
Lys57A proton donor

Chemical Components

hydrolysis, proton transfer, ingold: unimolecular elimination by the conjugate base

Step 5. Lys57 acts as a nucleophile towards the PLP-serine complex, forming a precursor to the regeneration of the internal aldimine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys57A covalently attached
Ser82A electrostatic stabiliser
Ser308A electrostatic stabiliser
Glu208A metal ligand
Gly212A (main-C) metal ligand
Asp214A metal ligand
Lys57A proton donor, nucleophile

Chemical Components

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

Step 6. Lys57 eliminates the enzymatic product to generate the reactive cofactor species which is capable of performing a new catalytic cycle.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys57A covalently attached
Ser82A electrostatic stabiliser
Ser308A electrostatic stabiliser
Glu208A metal ligand
Gly212A (main-C) metal ligand
Asp214A metal ligand
Lys57A electron pair donor

Chemical Components

elimination (not covered by the Ingold mechanisms), native state of cofactor regenerated, native state of enzyme regenerated

Step 7. Hydrolysis of the 2-aminoacrylate enzymatic product into pyruvate and ammonia is thought to occur outside the enzyme active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

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

Step 8. Final step of the non-enzymatic hydrolysis.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, ingold: unimolecular elimination by the conjugate base, overall product formed
Download: Image, Marvin File

Step 1. The neutral amine group of L-serine attacks the imine functionality of the pyridoxal-5-phosphate cofactor, forming a Schiff base precursor.

Step 2. The tetrahedral intermediate collapses, generating the external aldimine, PDD-substrate complex.

Step 3. Lys57 acts as a general base towards the C-alpha of the covalently bound serine, forming a planar sterocentre [PMID:19640845].

Step 4. Beta-elimination of water from the carbaion can occur to form an amino-acrylate intermediate.

Step 5. Lys57 acts as a nucleophile towards the PLP-serine complex, forming a precursor to the regeneration of the internal aldimine.

Step 6. Lys57 eliminates the enzymatic product to generate the reactive cofactor species which is capable of performing a new catalytic cycle.

Step 7. Hydrolysis of the 2-aminoacrylate enzymatic product into pyruvate and ammonia is thought to occur outside the enzyme active site.

Step 8. Final step of the non-enzymatic hydrolysis.

Products.

Introduction

This alternative mechanism is the same as the other proposal except for the elimination of water occurring via quinonoid intermediate rather than a carbanionic intermediate. However the carbanionic mechanism seems more likely to be correct, as it is is argued: "that both the si-face lysine ammonium ion and a reface serine residue are of central importance is stabilizing this key mechanistic intermediate." See other mechanism for the other references.

Catalytic Residues Roles

UniProt PDB* (2zr8)
*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

schiff base formed, overall reactant used, bimolecular nucleophilic addition, proton transfer, enzyme-substrate complex cleavage, unimolecular elimination by the conjugate base, electron transfer, dehydration, enzyme-substrate complex formation, native state of enzyme regenerated, native state of cofactor regenerated, elimination (not covered by the Ingold mechanisms), reaction occurs outside the enzyme, overall product formed

References

  1. Nelson DL et al. (2017), J Biol Chem, 292, 13986-14002. Human serine racemase structure/activity relationship studies provide mechanistic insight and point to position 84 as a hot spot for β-elimination function. DOI:10.1074/jbc.M117.777904. PMID:28696262.

Step 1. The neutral amine group of L-serine attacks the imine functionality of the pyridoxal-5-phosphate cofactor, forming a Schiff base precursor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser308A electrostatic stabiliser
Ser82A electrostatic stabiliser
Lys57A covalently attached
Asp214A metal ligand
Gly212A (main-C) metal ligand
Glu208A metal ligand
Lys57A proton acceptor, electron pair acceptor

Chemical Components

schiff base formed, overall reactant used, ingold: bimolecular nucleophilic addition, proton transfer

Step 2. The tetrahedral intermediate collapses, generating the external aldimine, PDD-substrate complex.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser308A electrostatic stabiliser
Ser82A electrostatic stabiliser
Lys57A covalently attached
Asp214A metal ligand
Gly212A (main-C) metal ligand
Glu208A metal ligand
Lys57A nucleofuge

Chemical Components

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

Step 3. Lys57 acts as a general base towards the C-alpha of the covalently bound serine. Deprotonation is facilitated by the aromatic ring acting as an electron sink.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser82A electrostatic stabiliser
Ser308A electrostatic stabiliser
Lys57A proton acceptor

Chemical Components

proton transfer, electron transfer

Step 4. Electron movement within the quinonoid intermediate facilitates the elimination of water. Lys57 now acting as an acid donates a proton to the hydroxyl promoting its elimination as water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser82A electrostatic stabiliser
Ser308A electrostatic stabiliser
Lys57A proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, dehydration, electron transfer

Step 5. Lys57 acts as a nucleophile towards the PLP-serine complex, forming a precursor to the regeneration of the internal aldimine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp214A metal ligand
Gly212A (main-C) metal ligand
Glu208A metal ligand
Ser308A electrostatic stabiliser
Ser82A electrostatic stabiliser
Lys57A covalently attached
Lys57A nucleophile, proton donor

Chemical Components

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

Step 6. Lys57 eliminates the enzymatic product to generate the reactive cofactor species which is capable of performing a new catalytic cycle.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp214A metal ligand
Gly212A (main-C) metal ligand
Glu208A metal ligand
Ser308A electrostatic stabiliser
Ser82A electrostatic stabiliser
Lys57A covalently attached, electron pair donor

Chemical Components

native state of enzyme regenerated, native state of cofactor regenerated, elimination (not covered by the Ingold mechanisms)

Step 7. Hydrolysis of the 2-aminoacrylate enzymatic product into pyruvate and ammonia is thought to occur outside the enzyme active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

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

Step 8. Final step of the non-enzymatic hydrolysis.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

overall product formed, ingold: unimolecular elimination by the conjugate base, reaction occurs outside the enzyme
Download: Image, Marvin File

Step 1. The neutral amine group of L-serine attacks the imine functionality of the pyridoxal-5-phosphate cofactor, forming a Schiff base precursor.

Step 2. The tetrahedral intermediate collapses, generating the external aldimine, PDD-substrate complex.

Step 3. Lys57 acts as a general base towards the C-alpha of the covalently bound serine. Deprotonation is facilitated by the aromatic ring acting as an electron sink.

Step 4. Electron movement within the quinonoid intermediate facilitates the elimination of water. Lys57 now acting as an acid donates a proton to the hydroxyl promoting its elimination as water.

Step 5. Lys57 acts as a nucleophile towards the PLP-serine complex, forming a precursor to the regeneration of the internal aldimine.

Step 6. Lys57 eliminates the enzymatic product to generate the reactive cofactor species which is capable of performing a new catalytic cycle.

Step 7. Hydrolysis of the 2-aminoacrylate enzymatic product into pyruvate and ammonia is thought to occur outside the enzyme active site.

Step 8. Final step of the non-enzymatic hydrolysis.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, James Willey