Phosphoenolpyruvate mutase
Phosphoenolpyruvate (PEP) mutase catalyses the conversion of PEP to 3-phosphonopyruvate which is the P-C bond forming step of the biosynthetic pathways leading to phosphonate natural products. Phosphonates are secondary metabolites, some of which have antibiotic activity. The structure of the enzyme is that of a modified alpha-beta barrel with only 7 beta sheets in the core of the barrel.
Reference Protein and Structure
- Sequence
-
P56839
(5.4.2.9)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Mytilus edulis (Blue mussel)
- PDB
-
1pym
- PHOSPHOENOLPYRUVATE MUTASE FROM MOLLUSK IN WITH BOUND MG2-OXALATE
(1.8 Å)
- Catalytic CATH Domains
-
3.20.20.60
(see all for 1pym)
- Cofactors
- Magnesium(2+) (1) Metal MACiE
Enzyme Reaction (EC:5.4.2.9)
+
→
Alternative enzyme names: PEP phosphomutase, PEPPM, Phosphoenolpyruvate phosphomutase, Phosphoenolpyruvate-phosphonopyruvate phosphomutase, PEP mutase,
Enzyme Mechanism
Introduction
This mechanism proposal represents the dissociative mechanism with a trigonal metaphosphate intermediate. Here the metaphosphate dissociates from the phosphoenolpyruvate substrate. Followed by an oxyanion initiated a nulceophilic attack of the C3 on the metaphosphate to form the product.
Catalytic Residues Roles
| UniProt | PDB* (1pym) | ||
| Ser46, Leu48 (main-N), Gly47 (main-N) | Ser46A, Leu48A (main-N), Gly47A (main-N) | Bind the substrate in the correct orientation. | hydrogen bond donor, electrostatic stabiliser, steric role |
| Arg159, His190, Asn122, Ser123, Lys120 | Arg159A, His190A, Asn122A, Ser123A, Lys120A | Act to stabilise the reactive intermediates, binding the phosphate group and activating it for heterolysis. | promote heterolysis, hydrogen bond donor, electrostatic stabiliser |
| Asp58 | Asp58A | The initial proposal had Asp58 acting as the phosphoryl carrier [PMID:10378273]. However, subsequent studies have suggested that the disassociative mechanism is more likely in which Asp58 helps activate the phosphate for disassociation from the substrate. | promote heterolysis, metal ligand |
| Asp85, Glu114, Asp87, Asp58 | Asp85A, Glu114A, Asp87A, Asp58A | Coordinate water molecules which coordinate the Mg ion, which stabilizes the reactive intermediates. | hydrogen bond donor, metal ligand, 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
heterolysis, intermediate formation, overall reactant used, bimolecular nucleophilic addition, intermediate terminated, overall product formed, native state of enzyme regeneratedReferences
- Xu D et al. (2005), J Phys Chem B, 109, 13827-13834. Theoretical Studies of Dissociative Phosphoryl Transfer in Interconversion of Phosphoenolpyruvate to Phosphonopyruvate: Solvent Effects, Thio Effects, and Implications for Enzymatic Reactions. DOI:10.1021/jp051042i. PMID:16852731.
- Xu D et al. (2008), J Phys Chem B, 112, 4102-4108. Ab Initio QM/MM Studies of the Phosphoryl Transfer Reaction Catalyzed by PEP Mutase Suggest a Dissociative Metaphosphate Transition State. DOI:10.1021/jp0776816. PMID:18331021.
- Liu S et al. (2002), Biochemistry, 41, 10270-10276. Dissociative Phosphoryl Transfer in PEP Mutase Catalysis: Structure of the Enzyme/Sulfopyruvate Complex and Kinetic Properties of Mutants†,‡. DOI:10.1021/bi026024v. PMID:12162742.
- Huang K et al. (1999), Structure, 7, 539-548. Helix swapping between two α/β barrels: crystal structure of phosphoenolpyruvate mutase with bound Mg2+–oxalate. DOI:10.1016/s0969-2126(99)80070-7. PMID:10378273.
Step 1. The metaphosphate dissociates from the phosphoenolpyruvate substrate.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Arg159A | hydrogen bond donor, electrostatic stabiliser, promote heterolysis |
| Ser46A | hydrogen bond donor, electrostatic stabiliser, steric role |
| Leu48A (main-N) | hydrogen bond donor, electrostatic stabiliser, steric role |
| Ser123A | hydrogen bond donor, electrostatic stabiliser, promote heterolysis |
| Asn122A | hydrogen bond donor, electrostatic stabiliser, promote heterolysis |
| His190A | hydrogen bond donor, electrostatic stabiliser, promote heterolysis |
| Gly47A (main-N) | electrostatic stabiliser |
| Lys120A | electrostatic stabiliser |
| Asp58A | metal ligand, promote heterolysis |
| Lys120A | promote heterolysis |
| Gly47A (main-N) | steric role |
| Asp85A | metal ligand |
| Asp87A | electrostatic stabiliser |
| Glu114A | electrostatic stabiliser |
| Asp85A | hydrogen bond donor |
| Asp87A | hydrogen bond donor |
| Glu114A | hydrogen bond donor |
Chemical Components
heterolysis, intermediate formation, overall reactant used
Step 2. The oxyanion initiated a nulceophilic attack of the C3 on the metaphosphate to form the product. The metaphosphate's interactions hole is stationary, whilst the C1-C2 bond os the pyruvate enolate rotates so that the C3-P bond can be formed [PMID:12162742].
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Arg159A | hydrogen bond donor, electrostatic stabiliser |
| Ser46A | hydrogen bond donor, electrostatic stabiliser, steric role |
| Leu48A (main-N) | hydrogen bond donor, electrostatic stabiliser, steric role |
| Ser123A | hydrogen bond donor, electrostatic stabiliser |
| Asn122A | hydrogen bond donor, electrostatic stabiliser |
| His190A | hydrogen bond donor, electrostatic stabiliser |
| Gly47A (main-N) | electrostatic stabiliser |
| Lys120A | electrostatic stabiliser |
| Gly47A (main-N) | steric role |
| Asp85A | metal ligand |
| Asp85A | electrostatic stabiliser |
| Asp87A | electrostatic stabiliser |
| Glu114A | electrostatic stabiliser |
| Asp85A | hydrogen bond donor |
| Asp87A | hydrogen bond donor |
| Glu114A | hydrogen bond donor |
Chemical Components
ingold: bimolecular nucleophilic addition, intermediate terminated, overall product formed, native state of enzyme regenerated
Download: Introduction
This mechanism proposal represents the associative mechanism with a phosphorylated aspartate intermediate. Attack from Asp58 to the phosphate leads to a nucleophilic substitution reaction with oxyanion and phosphorylated aspartate intermediates being formed. This is followed by an oxyanion initiated a nulceophilic attack of the C3 on the phosphate to form the product in a second substitution reaction.
Catalytic Residues Roles
| UniProt | PDB* (1pym) | ||
| Ser46, Leu48 (main-N), Gly47 (main-N) | Ser46A, Leu48A (main-N), Gly47A (main-N) | Bind the substrate in the correct orientation. | hydrogen bond donor, electrostatic stabiliser, steric role |
| Arg159, His190, Asn122, Ser123, Lys120 | Arg159A, His190A, Asn122A, Ser123A, Lys120A | Act to stabilize the reactive intermediates and binding the phosphate group. | promote heterolysis, hydrogen bond donor, electrostatic stabiliser |
| Asp58 | Asp58A | Acts as the phosphoryl carrier | nucleofuge, nucleophile |
| Asp85, Glu114, Asp87, Asp58 | Asp85A, Glu114A, Asp87A, Asp58A | Coordinate water molecules which coordinate the Mg ion, which stabilizes the reactive intermediates. | hydrogen bond donor, metal ligand, 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
intermediate formation, overall reactant used, bimolecular nucleophilic substitution, intermediate terminated, overall product formed, native state of enzyme regeneratedReferences
- Huang K et al. (1999), Structure, 7, 539-548. Helix swapping between two α/β barrels: crystal structure of phosphoenolpyruvate mutase with bound Mg2+–oxalate. DOI:10.1016/s0969-2126(99)80070-7. PMID:10378273.
- Xu D et al. (2008), J Phys Chem B, 112, 4102-4108. Ab Initio QM/MM Studies of the Phosphoryl Transfer Reaction Catalyzed by PEP Mutase Suggest a Dissociative Metaphosphate Transition State. DOI:10.1021/jp0776816. PMID:18331021.
- Liu S et al. (2002), Biochemistry, 41, 10270-10276. Dissociative Phosphoryl Transfer in PEP Mutase Catalysis: Structure of the Enzyme/Sulfopyruvate Complex and Kinetic Properties of Mutants†,‡. DOI:10.1021/bi026024v. PMID:12162742.
Step 1. Asp58 attacks the phosphate in a nucleophillic substitution reaction, forming a phosphorylated aspartate intermediate.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Glu114A | electrostatic stabiliser |
| Asp87A | electrostatic stabiliser |
| Asp85A | metal ligand |
| Lys120A | promote heterolysis, electrostatic stabiliser |
| Gly47A (main-N) | electrostatic stabiliser, steric role |
| Arg159A | hydrogen bond donor, electrostatic stabiliser, promote heterolysis |
| Ser46A | hydrogen bond donor, electrostatic stabiliser, steric role |
| Leu48A (main-N) | hydrogen bond donor, electrostatic stabiliser, steric role |
| Ser123A | hydrogen bond donor, electrostatic stabiliser, promote heterolysis |
| Asn122A | hydrogen bond donor, electrostatic stabiliser, promote heterolysis |
| His190A | hydrogen bond donor, electrostatic stabiliser, promote heterolysis |
| Asp85A | hydrogen bond donor |
| Asp87A | hydrogen bond donor |
| Glu114A | hydrogen bond donor |
| Asp58A | nucleophile |
Chemical Components
intermediate formation, overall reactant used, ingold: bimolecular nucleophilic substitution
Step 2. There is nucelophilic attack from the C=C bond of the substrate to the Asp bound phosphate leading to the termination of the intermediate and the formation of the product.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Ser46A | electrostatic stabiliser |
| Gly47A (main-N) | electrostatic stabiliser |
| Leu48A (main-N) | electrostatic stabiliser |
| Asp85A | electrostatic stabiliser |
| Asp87A | electrostatic stabiliser |
| Glu114A | electrostatic stabiliser |
| Lys120A | electrostatic stabiliser |
| Asn122A | electrostatic stabiliser |
| Ser123A | electrostatic stabiliser |
| Arg159A | electrostatic stabiliser |
| His190A | electrostatic stabiliser |
| Asp85A | metal ligand |
| Ser46A | steric role |
| Gly47A (main-N) | steric role |
| Leu48A (main-N) | steric role |
| Ser46A | hydrogen bond donor |
| Leu48A (main-N) | hydrogen bond donor |
| Asp85A | hydrogen bond donor |
| Asp87A | hydrogen bond donor |
| Glu114A | hydrogen bond donor |
| Asn122A | hydrogen bond donor |
| Ser123A | hydrogen bond donor |
| Arg159A | hydrogen bond donor |
| Asp58A | nucleofuge |
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