Fructose-bisphosphatase

 

Fructose 1,6-bisphosphatase (FBPase) hydrolyses fructose 1,6-bis-phosphate to fructose 6-phosphate and phosphate. The enzyme plays a crucial role in gluconeogenesis, the formation of glucose from non-carbohydrate carbon containing substrates. The enzyme is only capable of catalysing the forward reaction, while phosphofructokinase catalyses the reverse reaction. Regulation of the two enzyme is performed by metabolites such as fructose 2,6 bis-phosphate, ensuring that enhances reactivity of one enzyme is accompanied by suppressed reactivity of the other.

 

Reference Protein and Structure

Sequence
P00636 UniProt (3.1.3.11) IPR028343 (Sequence Homologues) (PDB Homologues)
Biological species
Sus scrofa (pig) Uniprot
PDB
1eyi - FRUCTOSE-1,6-BISPHOSPHATASE COMPLEX WITH MAGNESIUM, FRUCTOSE-6-PHOSPHATE AND PHOSPHATE (R-STATE) (2.32 Å) PDBe PDBsum 1eyi
Catalytic CATH Domains
3.30.540.10 CATHdb 3.40.190.80 CATHdb (see all for 1eyi)
Cofactors
Magnesium(2+) (3)
Click To Show Structure

Enzyme Reaction (EC:3.1.3.11)

water
CHEBI:15377ChEBI
+
D-fructofuranose 1,6-bisphosphate(4-)
CHEBI:49299ChEBI
→
hydrogenphosphate
CHEBI:43474ChEBI
+
beta-D-fructofuranose 6-phosphate(2-)
CHEBI:57634ChEBI
Alternative enzyme names: D-fructose 1,6-diphosphatase, D-fructose-1,6-bisphosphate phosphatase, FBPase, Fructose 1,6-bisphosphatase, Fructose 1,6-bisphosphate 1-phosphatase, Fructose 1,6-bisphosphate phosphatase, Fructose 1,6-diphosphatase, Fructose 1,6-diphosphate phosphatase, Fructose bisphosphate phosphatase, Fructose diphosphatase, Fructose diphosphate phosphatase, Hexose bisphosphatase, Hexose diphosphatase,

Enzyme Mechanism

Introduction

There are three divalent metal cations present within the catalytic site. Mg(2+) at site one is directly coordinated to the 1-(OH) of the 6 fructose phosphate product as a fifth coordinating ligand. The cation is thought to stabilise the negative charge of the hydroxide group before the transfer of a proton from Asp68, which relays a proton from the substrate neutralises the product. The second metal coordinates to a nucleophilic water molecule, also polarised through hydrogen bonds to Asp74 and Glu98, which abstracts a proton from a second coordinated water. The resulting hydroxide ion is the attacking nucleophile, displacing the 6 fructose phosphate in an SN2 mechanism. The resulting oxide is protonated as stated above.

Catalytic Residues Roles

UniProt PDB* (1eyi)
Glu98 Glu97A Acts as a bidentate ligand for Mg1 and Mg2 magnesium binding sites. metal ligand
Asp69 Asp68A The residue acts as a proton carrier from the substrate to the product. It initially forms a hydrogen bond with the proton of the 1-phosphate group, and after the SN2 displacement step has taken place, relays the proton to the anionic 6-fructose phosphate intermediate. metal ligand, proton acceptor, proton donor
Asp75 Asp74A Acts as the general acid/base that abstracts the proton from the catalytic water molecule. proton acceptor, electrostatic stabiliser, proton donor
Asp119 Asp118A Acts as a bidentate ligand for the Mg2 and Mg3 magnesium binding sites. metal ligand
Leu121 (main-C) Leu120A (main-C) Forms part of the Mg2 magnesium binding site. metal ligand
Asp122, Glu281 Asp121A, Glu280A Forms part of the Mg3 magnesium binding site. metal ligand
Glu99 Glu98A The residue's anionic carboxylic side chain hydrogen bonds to the basic water molecule in the close proximity Mg(2+) coordination sphere. The resulting polarisation enhances the basic character of the water molecule, activating it towards deprotonating a second, adjacent water molecule. 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

proton transfer, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Choe JY et al. (2000), Biochemistry, 39, 8565-8574. Crystal Structures of Fructose 1,6-Bisphosphatase:  Mechanism of Catalysis and Allosteric Inhibition Revealed in Product Complexes†,‡. DOI:10.1021/bi000574g. PMID:10913263.
  2. Gao Y et al. (2013), Biochemistry, 52, 5206-5216. Mechanism of displacement of a catalytically essential loop from the active site of mammalian fructose-1,6-bisphosphatase. DOI:10.1021/bi400532n. PMID:23844654.
  3. Iancu CV et al. (2005), J Biol Chem, 280, 19737-19745. R-state AMP complex reveals initial steps of the quaternary transition of fructose-1,6-bisphosphatase. DOI:10.1074/jbc.M501011200. PMID:15767255.
  4. Choe JY et al. (2003), J Biol Chem, 278, 16015-16020. Metaphosphate in the active site of fructose-1,6-bisphosphatase. DOI:10.1074/jbc.M212395200. PMID:12595528.
  5. Choe JY et al. (2003), J Biol Chem, 278, 16008-16014. Interaction of Tl+ with product complexes of fructose-1,6-bisphosphatase. DOI:10.1074/jbc.M212394200. PMID:12595529.

Step 1. Asp74 abstracts a proton from the catalytic water, which attacks the metal bound phosphate and eliminates the F6P as a negatively charged intermediate in an SN2-type reaction. It is likely that the reaction proceeds via a pentavalent transition state.

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Catalytic Residues Roles

Residue Roles
Asp74A electrostatic stabiliser
Glu98A electrostatic stabiliser
Asp118A metal ligand
Leu120A (main-C) metal ligand
Glu97A metal ligand
Asp68A metal ligand
Asp121A metal ligand
Glu280A metal ligand
Asp74A proton acceptor

Chemical Components

Step 2. Asp68 abstracts the proton from the phosphate group,

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp118A metal ligand
Leu120A (main-C) metal ligand
Glu97A metal ligand
Asp68A metal ligand
Asp121A metal ligand
Glu280A metal ligand
Glu98A electrostatic stabiliser
Asp68A proton acceptor

Chemical Components

proton transfer

Step 3. F6P abstracts the proton from Asp68 to generate the final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp118A metal ligand
Leu120A (main-C) metal ligand
Glu97A metal ligand
Asp68A metal ligand
Asp121A metal ligand
Glu280A metal ligand
Glu98A electrostatic stabiliser
Asp68A proton donor

Chemical Components

proton transfer, overall product formed

Step 4. Inferred return step. Asp74 returns a proton to water, which is then transferred to bulk solvent.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp118A metal ligand
Leu120A (main-C) metal ligand
Glu97A metal ligand
Asp68A metal ligand
Asp121A metal ligand
Glu280A metal ligand
Glu98A electrostatic stabiliser
Asp74A proton donor

Chemical Components

proton transfer, inferred reaction step, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Asp74 abstracts a proton from the catalytic water, which attacks the metal bound phosphate and eliminates the F6P as a negatively charged intermediate in an SN2-type reaction. It is likely that the reaction proceeds via a pentavalent transition state.

Step 2. Asp68 abstracts the proton from the phosphate group,

Step 3. F6P abstracts the proton from Asp68 to generate the final product.

Step 4. Inferred return step. Asp74 returns a proton to water, which is then transferred to bulk solvent.

Products.

Introduction

This represents the disassociative mechanism in which the P-O bond in fructose bisphosphate (FBP) breaks to form a postively charged phospho intermediate. Water is then activated by Asp74. The two reactive intermediates then combine for form a phosphate moiety.

Catalytic Residues Roles

UniProt PDB* (1eyi)
Glu98 Glu97A Acts as a bidentate ligand for Mg1 and Mg2 magnesium binding sites. metal ligand
Asp69 Asp68A The residue acts as a proton carrier from the substrate to the product. It initially forms a hydrogen bond with the proton of the 1-phosphate group, and after the displacement step has taken place, relays the proton to the anionic 6-fructose phosphate intermediate. metal ligand, proton acceptor, proton donor
Asp75 Asp74A Acts as a general acid/base that abstracts the proton from the catalytic water. proton acceptor, electrostatic stabiliser, proton donor
Asp119 Asp118A Acts as a bidentate ligand for the Mg2 and Mg3 magnesium binding sites. metal ligand
Leu121 (main-C) Leu120A (main-C) Forms part of the Mg2 magnesium binding site. metal ligand
Asp122, Glu281 Asp121A, Glu280A Forms part of the Mg3 magnesium binding site. metal ligand
Glu99 Glu98A The residue's anionic carboxylic side chain hydrogen bonds to the basic water molecule in the close proximity Mg(2+) coordination sphere. The resulting polarisation enhances the basic character of the water molecule, activating it towards deprotonating a second, adjacent water molecule. 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, overall reactant used, proton transfer, bimolecular nucleophilic addition, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Choe JY et al. (2003), J Biol Chem, 278, 16015-16020. Metaphosphate in the active site of fructose-1,6-bisphosphatase. DOI:10.1074/jbc.M212395200. PMID:12595528.

Step 1. The phsophate group dissociates from the FBP substrate to form the trigonal phosphate and negatively charged F6P intermediates.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu98A electrostatic stabiliser
Asp74A electrostatic stabiliser
Asp118A metal ligand
Leu120A (main-C) metal ligand
Glu97A metal ligand
Asp68A metal ligand
Asp121A metal ligand
Glu280A metal ligand

Chemical Components

heterolysis, overall reactant used

Step 2. Asp74 abstracts a proton from the catalytic water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp118A metal ligand
Leu120A (main-C) metal ligand
Glu97A metal ligand
Asp68A metal ligand
Asp121A metal ligand
Glu280A metal ligand
Glu98A electrostatic stabiliser
Asp74A proton acceptor

Chemical Components

proton transfer, overall reactant used

Step 3. The hydroxide attacks the positively charged phosphorous atom.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp118A metal ligand
Leu120A (main-C) metal ligand
Glu97A metal ligand
Asp68A metal ligand
Asp121A metal ligand
Glu280A metal ligand
Glu98A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, overall product formed

Step 4. The negatively charged F6P oxygen abstracts the proton from the phosphate group via Asp68. Not shown for clarity as it is assumed that this process is concerted.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp118A metal ligand
Leu120A (main-C) metal ligand
Glu97A metal ligand
Asp68A metal ligand
Asp121A metal ligand
Glu280A metal ligand
Glu98A electrostatic stabiliser
Asp68A proton acceptor

Chemical Components

proton transfer

Step 5. F6P abstracts the proton from Asp68 to generate the final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp118A metal ligand
Leu120A (main-C) metal ligand
Glu97A metal ligand
Asp68A metal ligand
Asp121A metal ligand
Glu280A metal ligand
Glu98A electrostatic stabiliser
Asp68A proton donor

Chemical Components

overall product formed, proton transfer

Step 6. Inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp118A metal ligand
Leu120A (main-C) metal ligand
Glu97A metal ligand
Asp68A metal ligand
Asp121A metal ligand
Glu280A metal ligand
Asp74A proton donor

Chemical Components

inferred reaction step, native state of enzyme regenerated, proton transfer
Download: Image, Marvin File

Step 1. The phsophate group dissociates from the FBP substrate to form the trigonal phosphate and negatively charged F6P intermediates.

Step 2. Asp74 abstracts a proton from the catalytic water molecule.

Step 3. The hydroxide attacks the positively charged phosphorous atom.

Step 4. The negatively charged F6P oxygen abstracts the proton from the phosphate group via Asp68. Not shown for clarity as it is assumed that this process is concerted.

Step 5. F6P abstracts the proton from Asp68 to generate the final product.

Step 6. Inferred return step.

Products.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday