Fructose-2,6-bisphosphate 2-phosphatase

 

Fructose-6-phosphate-2-kinase/fructose-2,6-biphosphatase is a bifunctional enzyme. It is responsible to regulate the concentration of fructose-2,6-phosphate (Fru-2,6-P2), a potent physiological activator of phosphofructose kinase and an inhibitor of fructose-1,6-biphosphatase. Therefore, Fru-6-P,2-kinase/Fru-2,6-Pase is important in glucose homeostasis. The fructose-2,6-biphosphatase domain has been shown to be structurally and functionally homologous to phosphoglycerate mutase. It catalyses its reaction via a phosphoenzyme intermediate which utilises an active site histidine as a nucleophilic phosphoacceptor.

 

Reference Protein and Structure

Sequence
P25114 UniProt (2.7.1.105, 3.1.3.46) IPR003094 (Sequence Homologues) (PDB Homologues)
Biological species
Rattus norvegicus (Norway rat) Uniprot
PDB
2bif - 6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE H256A MUTANT WITH F6P IN PHOSPHATASE ACTIVE SITE (2.4 Å) PDBe PDBsum 2bif
Catalytic CATH Domains
3.40.50.1240 CATHdb (see all for 2bif)
Click To Show Structure

Enzyme Reaction (EC:3.1.3.46)

beta-D-fructofuranose 2,6-bisphosphate(4-)
CHEBI:58579ChEBI
+
water
CHEBI:15377ChEBI
→
beta-D-fructofuranose 6-phosphate(2-)
CHEBI:57634ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: D-fructose-2,6-bisphosphate 2-phosphohydrolase, Fructose-2,6-bisphosphatase,

Enzyme Mechanism

Introduction

The catalytic reaction proceeds in two steps: (1) Formation of the phosphoryl-histidine intermediate and release of fructose 6-phosphate; and (2) hydrolysis of the phosphoenzyme intermediate. The proposed mechanism has His236 acting as a nucleophile to attack the phosphate, leading to a pentacovalent transition state, where the excess negative charge is stabilised by Arg255, Asn262, Arg305 and His390. A protonated Glu325 donates a proton to the leaving Fru-6-P. Ionised Glu325 activates a water nucleophile to attack the phosphohistidine intermediate and restore the enzyme.

Catalytic Residues Roles

UniProt PDB* (2bif)
His257 Ala256(257)A It acts as a nucleophile to attack the target phosphate of the substrate, forming a phosphoenzyme intermediate. covalently attached, nucleofuge, nucleophile
Arg306 Arg305(306)A It neutralises the excess negative charge developed on the phosphate of Fru-2,6-P2 during the transfer to His 256, hence stabilises the transition state. electrostatic stabiliser
Glu326 Glu325(326)A It acts as an acid to protonate the leaving Fru-6-P and acts as a base to activate a water nucleophile, which attack the phosphohistidine intermediate to restore the enzyme. activator, proton acceptor, proton donor
Asn263, His391 Asn262(263)A, His390(391)A It interacts with the 2-phosphate of Fru-2,6-P2 and stabilises the transition state. electrostatic stabiliser
Arg256 Arg255(256)A It stabilises the transition state. 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, intermediate formation, overall product formed, enzyme-substrate complex formation, proton transfer, intermediate collapse, aromatic unimolecular elimination by the conjugate base, heterolysis, intermediate terminated, native state of enzyme regenerated, rate-determining step, enzyme-substrate complex cleavage

References

  1. Yuen MH et al. (1999), J Biol Chem, 274, 2176-2184. Crystal Structure of the H256A Mutant of Rat Testis Fructose-6-phosphate,2-kinase/Fructose-2,6-bisphosphatase: FRUCTOSE 6-PHOSPHATE IN THE ACTIVE SITE LEADS TO MECHANISMS FOR BOTH MUTANT AND WILD TYPE BISPHOSPHATASE ACTIVITIES. DOI:10.1074/jbc.274.4.2176. PMID:9890980.
  2. Sakurai M et al. (2000), Biochemistry, 39, 16238-16243. Glutamate 325 Is a General Acid−Base Catalyst in the Reaction Catalyzed by Fructose-2,6-bisphosphatase†. DOI:10.1021/bi0020170. PMID:11123954.
  3. Lin K et al. (1992), J Biol Chem, 267, 19163-19171. Arg-257 and Arg-307 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase bind the C-2 phospho group of fructose-2,6-bisphosphate in the fructose-2,6-bisphosphatase domain. PMID:1326547.
  4. Tauler A et al. (1990), J Biol Chem, 265, 15617-15622. Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Use of site-directed mutagenesis to evaluate the roles of His-258 and His-392 in catalysis. PMID:2168419.
  5. Pilkis SJ et al. (1984), J Biol Chem, 259, 949-958. Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation. PMID:6319392.
  6. Pilkis SJ et al. (1983), J Biol Chem, 258, 6135-6141. 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase from rat liver. PMID:6304027.

Step 1. Nucleophilic attack of 2-phosphate by His256 resulting in a tetrahedral intermediate which is stabilised by Arg205, Arg255, His390 and Asn262.

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Catalytic Residues Roles

Residue Roles
Asn262(263)A electrostatic stabiliser
His390(391)A electrostatic stabiliser
Arg255(256)A electrostatic stabiliser
Arg305(306)A electrostatic stabiliser
Ala256(257)A covalently attached, nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation, overall product formed, enzyme-substrate complex formation

Step 2. Fructose-6-phosphate is protonated by Glu325 and then exits the active site.

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Catalytic Residues Roles

Residue Roles
Ala256(257)A covalently attached
Glu325(326)A proton donor

Chemical Components

proton transfer, intermediate collapse, ingold: aromatic unimolecular elimination by the conjugate base, overall product formed

Step 3. Glu325 abstracts a proton off the water molecule, causing it to act as a nucleophile and attack phospho-His256. The tetrahedral intermediate formed is also stabilised by Arg305, Arg255, His390 and Asn262.

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Catalytic Residues Roles

Residue Roles
Arg255(256)A electrostatic stabiliser
Asn262(263)A electrostatic stabiliser
Arg305(306)A electrostatic stabiliser
His390(391)A electrostatic stabiliser
Glu325(326)A activator
Ala256(257)A covalently attached
Glu325(326)A proton acceptor

Chemical Components

intermediate formation, ingold: bimolecular nucleophilic addition, intermediate collapse

Step 4. The intermediate collapses, resulting in the cleavage of the phospho-His256 bond. The phosphate group is held in the active site until displaced by an incoming substrate.

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Catalytic Residues Roles

Residue Roles
Ala256(257)A nucleofuge

Chemical Components

heterolysis, intermediate terminated, native state of enzyme regenerated, overall product formed, rate-determining step, enzyme-substrate complex cleavage
Download: Image, Marvin File

Step 1. Nucleophilic attack of 2-phosphate by His256 resulting in a tetrahedral intermediate which is stabilised by Arg205, Arg255, His390 and Asn262.

Step 2. Fructose-6-phosphate is protonated by Glu325 and then exits the active site.

Step 3. Glu325 abstracts a proton off the water molecule, causing it to act as a nucleophile and attack phospho-His256. The tetrahedral intermediate formed is also stabilised by Arg305, Arg255, His390 and Asn262.

Step 4. The intermediate collapses, resulting in the cleavage of the phospho-His256 bond. The phosphate group is held in the active site until displaced by an incoming substrate.

Products.

Contributors

Mei Leung, Gemma L. Holliday, Morwenna Hall