Phosphoenolpyruvate carboxylase

 

Phosphoenolpyruvate carboxylase (PEPC) catalyses the irreversible carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate (OAA) and Pi using divalent Mg(II) or Mn(II) as a cofactor. The active species for initial carboxylation is not carbon dioxide but the chemically less reactive bicarbonate anion, which dominates in the cytoplasm where PEPC functions. The enzyme is present in all photosynthetic organisms, including higher plants, green algae, cyanobacteria and photosynthetic bacteria, and most nonphotosynthetic bacteria and protozoa, but is notably absent from animals, fungi, and yeasts. PEPC primarily plays an anaplerotic role by replenishing C4-dicarboxylic acids utilised for both energy and biosynthetic metabolisms.

 

Reference Protein and Structure

Sequence
P00864 UniProt (4.1.1.31) IPR022805 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1qb4 - CRYSTAL STRUCTURE OF MN(2+)-BOUND PHOSPHOENOLPYRUVATE CARBOXYLASE (2.6 Å) PDBe PDBsum 1qb4
Catalytic CATH Domains
(see all for 1qb4)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:4.1.1.31)

water
CHEBI:15377ChEBI
+
phosphonatoenolpyruvate
CHEBI:58702ChEBI
+
carbon dioxide
CHEBI:16526ChEBI
→
hydron
CHEBI:15378ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
+
oxaloacetate(2-)
CHEBI:16452ChEBI
Alternative enzyme names: PEP carboxylase, PEPC, PEPCase, Phosphoenolpyruvic carboxylase, Phosphopyruvate (phosphate) carboxylase, Phosphate:oxaloacetate carboxy-lyase (phosphorylating),

Enzyme Mechanism

Introduction

The first chemical step in the proposed mechanism is the nucleophilic attack by bicarbonate to form carboxyphosphate and the enolate of pyruvate. In order to make the phosphorus atom less negative, the positively charged electrostatic pocket formed by three arginine residues allows the dissipation of the negative charges of the phosphate group during the approach. In the second chemical step of the proposed mechanism His138 acts as a base to abstract a proton from carboxyphosphate; CO2 is generated, the hydrophobic environment in the active site pocket may stabilise the CO2 liberated from the carboxyphosphate. Finally the carbon dioxide generated is nucleophilically attacked by enolate of pyruvate to generate oxaloacetate. The phosphate anion abstracts a proton from His138 to form a phosphate group.

Catalytic Residues Roles

UniProt PDB* (1qb4)
Glu506, Asp543 Glu506A, Asp543A Coordinate the stabilizing metal metal ligand
His138 His138A Acts as a general acid/base. proton acceptor, proton donor
Arg581, Arg713, Arg396 Arg581A, Arg713A, Arg396A The positively charged electrostatic pocket formed by Arg396, Arg699, and Arg713 allows the dissipation of the negative charges of the phosphate group during the approach 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, bimolecular nucleophilic substitution, decarboxylation, aldol addition, overall product formed, native state of enzyme regenerated

References

  1. Matsumura H et al. (2002), Structure, 10, 1721-1730. Crystal structures of C4 form maize and quaternary complex of E. coli phosphoenolpyruvate carboxylases. PMID:12467579.
  2. Kai Y et al. (2003), Arch Biochem Biophys, 414, 170-179. Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms. PMID:12781768.
  3. Terada K et al. (1991), Eur J Biochem, 202, 797-803. Site-directed mutagenesis of the conserved histidine residue of phosphoenolpyruvate carboxylase. His138 is essential for the second partial reaction. PMID:1765093.

Step 1. There is nucleophilic attack from the water molecule to the CO2 molecule forming carbonicacid

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg396A electrostatic stabiliser
Arg581A electrostatic stabiliser
Arg713A electrostatic stabiliser
Glu506A metal ligand
Asp543A metal ligand

Chemical Components

ingold: bimolecular nucleophilic addition

Step 2. The carbonic acid is deprotonated, forming a bicarbonate ion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg396A electrostatic stabiliser
Arg581A electrostatic stabiliser
Arg713A electrostatic stabiliser
Glu506A metal ligand
Asp543A metal ligand

Chemical Components

proton transfer

Step 3. Nucleophilic attack by bicarbonate forms carboxyphosphate and the enolate of pyruvate. In order to make the phosphorus atom less negative, the positively charged electrostatic pocket formed by the arginine residues allows the dissipation of the negative charges of the phosphate group during the approach

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg396A electrostatic stabiliser
Arg581A electrostatic stabiliser
Arg713A electrostatic stabiliser
Glu506A metal ligand
Asp543A metal ligand

Chemical Components

overall reactant used, ingold: bimolecular nucleophilic substitution

Step 4. His 138 acts as a general base abstracting a proton from the carboxyphosphate, this leads to CO2 being released.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg396A electrostatic stabiliser
Arg581A electrostatic stabiliser
Arg713A electrostatic stabiliser
Glu506A metal ligand
Asp543A metal ligand
His138A proton acceptor

Chemical Components

decarboxylation, proton transfer

Step 5. An aldol addition leads to the phosphoenolpyruvate being carboxylated. The phosphate accepts a proton from His 138.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg396A electrostatic stabiliser
Arg581A electrostatic stabiliser
Arg713A electrostatic stabiliser
Glu506A metal ligand
Asp543A metal ligand
His138A proton donor

Chemical Components

aldol addition, overall product formed, proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. There is nucleophilic attack from the water molecule to the CO2 molecule forming carbonicacid

Step 2. The carbonic acid is deprotonated, forming a bicarbonate ion.

Step 3. Nucleophilic attack by bicarbonate forms carboxyphosphate and the enolate of pyruvate. In order to make the phosphorus atom less negative, the positively charged electrostatic pocket formed by the arginine residues allows the dissipation of the negative charges of the phosphate group during the approach

Step 4. His 138 acts as a general base abstracting a proton from the carboxyphosphate, this leads to CO2 being released.

Step 5. An aldol addition leads to the phosphoenolpyruvate being carboxylated. The phosphate accepts a proton from His 138.

Products.

Contributors

Anna Waters, Craig Porter, Gemma L. Holliday, James Willey