Diphosphate---fructose-6-phosphate 1-phosphotransferase

 

The Lyme disease causing Spirochete Borrelia burgdorferi uses pyrophosphate (PPi) rather than ATP as the source of phosphate for the phosphorylation of Fructose-6-phosphate to Fructose-1,6-bisphosphate in glycolysis. This strategy, common to many anaerobic bacteria, allows ATP to be conserved for other processes such as maintaining a proton gradient. The enzyme catalysing this process shares around 23% sequence identity to the E. coli PFK and many structural features differ between the two enzymes. However, the active sites do share a similar structure with conservation of key residues, so the mechanism of the reaction is almost certainly the same for each.

 

Reference Protein and Structure

Sequence
P70826 UniProt (2.7.1.90) IPR011183 (Sequence Homologues) (PDB Homologues)
Biological species
Borrelia burgdorferi B31 (Bacteria) Uniprot
PDB
1kzh - Structure of a pyrophosphate-dependent phosphofructokinase from the Lyme disease spirochete Borrelia burgdorferi (2.55 Å) PDBe PDBsum 1kzh
Catalytic CATH Domains
3.40.50.450 CATHdb (see all for 1kzh)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.7.1.90)

diphosphate(4-)
CHEBI:18361ChEBI
+
beta-D-fructofuranose 6-phosphate(2-)
CHEBI:57634ChEBI
→
hydrogenphosphate
CHEBI:43474ChEBI
+
D-fructofuranose 1,6-bisphosphate(4-)
CHEBI:49299ChEBI
Alternative enzyme names: 6-phosphofructokinase (pyrophosphate), Inorganic pyrophosphate-dependent phosphofructokinase, Inorganic pyrophosphate-phosphofructokinase, Pyrophosphate-dependent phosphofructo-1-kinase, Pyrophosphate--fructose 6-phosphate 1-phosphotransferase, Pyrophosphate--fructose 6-phosphate phosphotransferase, Diphosphate-dependent 6-phosphofructose-1-kinase, Pyrophosphate-dependent 6-phosphofructose-1-kinase, 6-phosphofructokinase (diphosphate),

Enzyme Mechanism

Introduction

Fructose-6-phosphate's 1COH acts as the nucleophile to attack the terminal phosphate of PPi. Its nucleophilicity is increased by the action of Asp 206 which removes a hydrogen ion from the attacking hydroxyl, allowing a pentavalent phosphate transition state to form, stabilised by Mg2+ and several conserved residues at the active site (Gly 82, Arg 146, Thr 204 and Lys 203). Collapse of the transition state results in the overall phosphoryl transferfrom PPi to fructose-6-phosphate. It is still unclear the exact location and coordination of the magnesium ion due to not being present in the crystal structure.

Catalytic Residues Roles

UniProt PDB* (1kzh)
Asp177 Asp177A Important in substrate specificity for pyrophosphate (not ATP) as the phosphate donor, indirectly coordinating to the magnesium cofactor via a water molecule. polar interaction
Gly82 (main-N) Gly82A (main-N) The amide on the polypeptide backbone forms a strong hydrogen bond to the pyrophosphate thus stabilising the pentavalent phosphate transition state. electrostatic stabiliser, polar interaction
Arg146 Arg146A Contacts PPi through its positively charged side chain, thus stabilises the pentavalent phosphate transition state. electrostatic stabiliser, polar interaction
Lys203 Lys203A Stabilises the pentavalent phosphate transition state through electrostatic interactions. electrostatic stabiliser, polar interaction
Thr204 Thr204A Forms hydrogen bond to the PPi, thus can act to stabilise the negative charge that develops in formation of the pentavalent phosphate transition state. electrostatic stabiliser, polar interaction
Asp206 Asp206A Activates the 1-OH group of fructose-6-phosphate to allow the nucleophilic attack on the terminal phosphate of PPi which leads to the in-line phosphoryl transfer. proton acceptor, proton donor, polar interaction, activator, 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 substitution, proton transfer, overall reactant used, overall product formed, inferred reaction step

References

  1. Moore SA et al. (2002), Structure, 10, 659-671. The structure of a pyrophosphate-dependent phosphofructokinase from the Lyme disease spirochete Borrelia burgdorferi. DOI:10.2210/pdb1kzh/pdb. PMID:12015149.

Step 1. Asp206 deprotonates Fructose-6-phosphate's 1C hydroxyl. This increases its nucleophilicity to then attack the terminal PPi phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr204A electrostatic stabiliser
Asp206A electrostatic stabiliser
Gly82A (main-N) electrostatic stabiliser
Lys203A electrostatic stabiliser
Arg146A electrostatic stabiliser
Asp206A activator
Gly82A (main-N) polar interaction
Arg146A polar interaction
Asp177A polar interaction
Lys203A polar interaction
Thr204A polar interaction
Asp206A polar interaction
Asp206A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, overall reactant used, overall product formed

Step 2. Proton transfer from Asp206 to the phosphate group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly82A (main-N) polar interaction
Arg146A polar interaction
Asp177A polar interaction
Lys203A polar interaction
Thr204A polar interaction
Asp206A polar interaction, proton donor

Chemical Components

inferred reaction step, proton transfer
Download: Image, Marvin File

Step 1. Asp206 deprotonates Fructose-6-phosphate's 1C hydroxyl. This increases its nucleophilicity to then attack the terminal PPi phosphate.

Step 2. Proton transfer from Asp206 to the phosphate group.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, Morwenna Hall