S00365
type: Enzyme
rlcp: p81
cath: Rossmann fold
ec: https://purl.uniprot.org/enzyme/3.1.3.46
csa: 2bif
uniprot: P07953
uniprot: Q9JJH5
uniprot: O35552
uniprot: P25114
kegg enzyme: fructose-2,6-bisphosphate 2-phosphatase
kegg enzyme: fructose-2,6-bisphosphatase
kegg enzyme: D-fructose-2,6-bisphosphate 2-phosphohydrolase
kegg pathway: Fructose and mannose metabolism
kegg substrate: D-Fructose 2,6-bisphosphate
kegg substrate: H2O
kegg product: D-Fructose 6-phosphate
kegg product: Orthophosphate
pdb: 1bifA02
pdb: 1fbtA
pdb: 1fbtB
pdb: 1tipA
pdb: 1tipB
pdb: 2bifA02
pdb: 2bifB02
pdb: 3bifA02
pdb bound state: Unbound
  compound: D-Fructose 2,6-bisphosphate
  pdb: 1bifA02
pdb bound state: Unbound
  compound: D-Fructose 2,6-bisphosphate
  pdb: 1fbtA
pdb bound state: Unbound
  compound: D-Fructose 2,6-bisphosphate
  pdb: 1fbtB
pdb bound state: Unbound
  compound: D-Fructose 2,6-bisphosphate
  pdb: 1tipA
pdb bound state: Unbound
  compound: D-Fructose 2,6-bisphosphate
  pdb: 1tipB
pdb bound state: Unbound
  compound: D-Fructose 2,6-bisphosphate
  pdb: 2bifA02
pdb bound state: Unbound
  compound: D-Fructose 2,6-bisphosphate
  pdb: 2bifB02
pdb bound state: Unbound
  compound: D-Fructose 2,6-bisphosphate
  pdb: 3bifA02
pdb bound state: Analogue
  compound: D-Fructose 6-phosphate
  pdb: 1bifA02
  ligand het code: PO4
pdb bound state: Unbound
  compound: D-Fructose 6-phosphate
  pdb: 1fbtA
pdb bound state: Unbound
  compound: D-Fructose 6-phosphate
  pdb: 1fbtB
pdb bound state: Analogue
  compound: D-Fructose 6-phosphate
  pdb: 3bifA02
  ligand het code: PO4
pdb bound state: Unbound
  compound: Orthophosphate
  pdb: 1tipA
pdb bound state: Unbound
  compound: Orthophosphate
  pdb: 1tipB
pdb bound state: Unbound
  pdb: 1bifA02
pdb bound state: Unbound
  pdb: 1fbtA
pdb bound state: Unbound
  pdb: 1fbtB
pdb bound state: Unbound
  pdb: 2bifA02
pdb bound state: Unbound
  pdb: 2bifB02
pdb bound state: Unbound
  pdb: 3bifA02
reference for catalytic mechanism: ReferenceForCatalyticMechanism
  reference index: 6
  reference section: Fig.4, p.6560-6561
  reference step: 3
reference for catalytic mechanism: ReferenceForCatalyticMechanism
  reference index: 8
  reference section: Fig.5, p.6015-6016
  reference step: 2
reference for catalytic mechanism: ReferenceForCatalyticMechanism
  reference index: 9
  reference section: Fig.5, p.1022-1024
  reference step: 2
reference for catalytic mechanism: ReferenceForCatalyticMechanism
  reference index: 10
  reference section: Fig.3, p.616-617
  reference step: 2
reference for catalytic mechanism: ReferenceForCatalyticMechanism
  reference index: 12
  reference section: p.4478
reference for catalytic mechanism: ReferenceForCatalyticMechanism
  reference index: 13
  reference section: Fig.7, p.2175
  reference step: 2
reference for catalytic mechanism: ReferenceForCatalyticMechanism
  reference index: 14
  reference section: Fig.5, p.2181-2183
  reference step: 3
reference for catalytic mechanism: ReferenceForCatalyticMechanism
  reference index: 15
  reference section: Fig.6, p.9759-9761
  reference step: 5
references: Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation.
references: Mechanism of activation of fructose-2,6-bisphosphatase by cAMP-dependent protein kinase.
references: Catalytic site of rat liver and bovine heart fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Identification of fructose 6-phosphate binding site.
references: Evolution of a bifunctional enzyme: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
references: Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties of phospho- and dephospho- forms and of two mutants in which Ser32 has been changed by site-directed mutagenesis.
references: Glu327 is part of a catalytic triad in rat liver fructose-2,6-bisphosphatase.
references: Identification of transient intermediates in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by 31P-NMR spectroscopy.
references: Crystal structure of the rat liver fructose-2,6-bisphosphatase based on selenomethionine multiwavelength anomalous dispersion phases.
references: The crystal structure of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase reveals distinct domain homologies.
references: Crystal structure of a trapped phosphoenzyme during a catalytic reaction.
references: Site-directed mutants of rat testis fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase: localization of conformational alterations induced by ligand binding.
references: http://ezcatdb.cbrc.jp/EzCatDB/reference/S00365#12
references: Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway.
references: http://ezcatdb.cbrc.jp/EzCatDB/reference/S00365#14
references: Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy.
references: The difference in the carboxy-terminal sequence is responsible for the difference in the activity of chicken and rat liver fructose-2,6-bisphosphatase.
references: Involvement of the chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase sequence His444-Arg-Glu-Arg in modulation of the bisphosphatase activity by its kinase domain.
comment: This enzyme belongs to the phosphoglycerate mutase family. There have been a number of proposed mechanisms for this phosphatase (see literature [6], [8], [9], [10], [12], [13], [14] & [15]). However, all of these proposed mechanisms are consistent in that the catalysis proceeds in the two steps, formation and hydrolysis (or breakdown) of the phosphohistidine intermediate. His256 (PDB 1bif) acts as a nucleophile, which attacks on the phosphorous atom of the target phosphate (2-phosphate of the substrate), forming the phosphohistidine intermediate. During the catalysis, the residues, Arg255, Asn262 and Arg305 (PDB 1bif), stabilize the transition state and the intermediate, by neutralizing the negative charges at the phosphate oxygens. Moreover, Glu325 (PDB 1bif) is thought to act as a pKa modulator, according to the literature [6] and [9]. In the first step of the intermediate formation, a catalytic acid protonates to the leaving group (the product, Fructose 6-phosphate). In the second step, a catalytic base activates a bound water molecule, which hydrolyze the phosphohistidine intermediate. Which residue could be the catalytic acid and base has been debated to date. Both Glu327 and His392 (PDB 1bif) have been identified as the catalytic acid and as the catalytic base activating the water for the hydrolysis (see [6], [8], [9], [10], [12], [13], [14] & [15]). The literature [6] and [8] proposed that His392 acts as the proton donor, whilst Glu327 activates the water as a general base. In contrast, the paper [9] suggested His390 play a dual role as acid/base, whilst another paper [14] proposed that Glu327 acts as acid/base.

Rossmann fold
type: Cath
references: http://www.cathdb.info/version/latest/superfamily/3.40.50.1240
catalytic domain: index.html

Fructose and mannose metabolism
type: KeggPathway
references: KEGG:MAP00051

D-Fructose 2,6-bisphosphate
type: KeggSubstrate
references: KEGG:C00665
compound type: carbohydrate
compound type: phosphate group/phosphate ion
references: CHEBI:28602
references: http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi

H2O
type: KeggSubstrate
references: KEGG:C00001
compound type: H2O
references: CHEBI:15377
references: http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi

D-Fructose 6-phosphate
type: KeggProduct
references: KEGG:C00085
compound type: carbohydrate
compound type: phosphate group/phosphate ion
references: CHEBI:61553
references: http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi

Orthophosphate
type: KeggProduct
references: KEGG:C00009
compound type: phosphate group/phosphate ion
references: CHEBI:26078
references: http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi

1bifA02
type: Pdb
references: 1bif
references: http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl
references: PDB:1bif
catalytic residue: GLU 325;HIS 390
modified residue: HIS 256

1fbtA
type: Pdb
references: 1fbt
references: http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl
references: PDB:1fbt
catalytic residue: GLU 77;HIS 142
modified residue: HIS 8

1fbtB
type: Pdb
references: 1fbt
references: http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl
references: PDB:1fbt
catalytic residue: GLU 77;HIS 142
modified residue: HIS 8

1tipA
type: Pdb
references: 1tip
references: http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl
references: PDB:1tip
catalytic residue: GLU 78;HIS 143
modified residue: NEP 9
comment: NEP (phosphorylated HIS)

1tipB
type: Pdb
references: 1tip
references: http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl
references: PDB:1tip
catalytic residue: GLU 78;HIS 143
modified residue: NEP 9
comment: NEP (phosphorylated HIS)

2bifA02
type: Pdb
references: 2bif
references: http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl
references: PDB:2bif
catalytic residue: GLU 325;HIS 390
comment: mutant H256A

2bifB02
type: Pdb
references: 2bif
references: http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl
references: PDB:2bif
catalytic residue: GLU 325;HIS 390
comment: mutant H256A

3bifA02
type: Pdb
references: 3bif
references: http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl
references: PDB:3bif
catalytic residue: GLU 325;HIS 390
modified residue: HIS 256

Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation.
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/6319392
journal: J Biol Chem
volume: 259(2)
pages: 949-58
published year: 1984
authors:
  item: Pilkis SJ
  item: Regen DM
  item: Stewart HB
  item: Pilkis J
  item: Pate TM
  item: El-Maghrabi MR

Mechanism of activation of fructose-2,6-bisphosphatase by cAMP-dependent protein kinase.
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/3013863
journal: J Biol Chem
volume: 261(19)
pages: 8793-8
published year: 1986
authors:
  item: Stewart HB
  item: el-Maghrabi MR
  item: Pilkis SJ

Catalytic site of rat liver and bovine heart fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Identification of fructose 6-phosphate binding site.
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/2539378
journal: J Biol Chem
volume: 264(11)
pages: 6344-8
published year: 1989
authors:
  item: Kitamura K
  item: Uyeda K
  item: Hartman FC
  item: Kangawa K
  item: Matsuo H
references: P07953

Evolution of a bifunctional enzyme: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/2557623
journal: Proc Natl Acad Sci U S A
volume: 86(24)
pages: 9642-6
published year: 1989
authors:
  item: Bazan JF
  item: Fletterick RJ
  item: Pilkis SJ

Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties of phospho- and dephospho- forms and of two mutants in which Ser32 has been changed by site-directed mutagenesis.
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/1339450
journal: J Biol Chem
volume: 267(7)
pages: 4416-23
published year: 1992
authors:
  item: Kurland IJ
  item: el-Maghrabi MR
  item: Correia JJ
  item: Pilkis SJ

Glu327 is part of a catalytic triad in rat liver fructose-2,6-bisphosphatase.
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/1313012
journal: J Biol Chem
volume: 267(10)
pages: 6556-62
published year: 1992
authors:
  item: Lin K
  item: Li L
  item: Correia JJ
  item: Pilkis SJ

Identification of transient intermediates in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by 31P-NMR spectroscopy.
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/7755565
journal: Biochem J
volume: 308 (Pt 1)
pages: 189-95
published year: 1995
authors:
  item: Okar DA
  item: Kakalis LT
  item: Narula SS
  item: Armitage IM
  item: Pilkis SJ

Crystal structure of the rat liver fructose-2,6-bisphosphatase based on selenomethionine multiwavelength anomalous dispersion phases.
type: BibliographicResource
comment: X-ray crystallography (2.5 Angstroms)
references: http://www.ncbi.nlm.nih.gov/pubmed/8634242
journal: Biochemistry
volume: 35(19)
pages: 6010-6019
published year: 1996
authors:
  item: Lee YH
  item: Ogata C
  item: Pflugrath JW
  item: Levitt DG
  item: Sarma R
  item: Banaszak LJ
  item: Pilkis SJ
references: http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl

The crystal structure of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase reveals distinct domain homologies.
type: BibliographicResource
comment: X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS)
references: http://www.ncbi.nlm.nih.gov/pubmed/8805587
journal: Structure
volume: 4(9)
pages: 1017-29
published year: 1996
authors:
  item: Hasemann CA
  item: Istvan ES
  item: Uyeda K
  item: Deisenhofer J
references: http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl
references: P25114

Crystal structure of a trapped phosphoenzyme during a catalytic reaction.
type: BibliographicResource
comment: X-ray crystallography
references: http://www.ncbi.nlm.nih.gov/pubmed/9253407
journal: Nat Struct Biol
volume: 4(8)
pages: 615-618
published year: 1997
authors:
  item: Lee YH
  item: Olson TW
  item: Ogata CM
  item: Levitt DG
  item: Banaszak LJ
  item: Lange AJ
references: http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl
references: P07953

Site-directed mutants of rat testis fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase: localization of conformational alterations induced by ligand binding.
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/9760241
journal: Biochemistry
volume: 37(40)
pages: 14057-64
published year: 1998
authors:
  item: Helms MK
  item: Hazlett TL
  item: Mizuguchi H
  item: Hasemann CA
  item: Uyeda K
  item: Jameson DM

http://ezcatdb.cbrc.jp/EzCatDB/reference/S00365#12
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/10194369
journal: Biochemistry
volume: 38(14)
pages: 4471-9
published year: 1999
authors:
  item: Okar DA
  item: Live DH
  item: Kirby TL
  item: Karschnia EJ
  item: von Weymarn LB
  item: Armitage IM
  item: Lange AJ
title: The roles of Glu-327 and His-446 in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by NMR spectroscopic and mutational analyses of the enzyme in the transient phosphohistidine intermediate complex.

Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway.
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/9890979
journal: J Biol Chem
volume: 274(4)
pages: 2166-75
published year: 1999
authors:
  item: Mizuguchi H
  item: Cook PF
  item: Tai CH
  item: Hasemann CA
  item: Uyeda K

http://ezcatdb.cbrc.jp/EzCatDB/reference/S00365#14
type: BibliographicResource
comment: X-RAY CRYSTALLOGRAPHY (2.4 ANGSTROMS) OF MUTANT ALA-256
references: http://www.ncbi.nlm.nih.gov/pubmed/9890980
journal: J Biol Chem
volume: 274(4)
pages: 2176-84
published year: 1999
authors:
  item: Yuen MH
  item: Mizuguchi H
  item: Lee YH
  item: Cook PF
  item: Uyeda K
  item: Hasemann CA
title: 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.
references: http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl
references: P25114

Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy.
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/10933792
journal: Biochemistry
volume: 39(32)
pages: 9754-62
published year: 2000
authors:
  item: Okar DA
  item: Live DH
  item: Devany MH
  item: Lange AJ

The difference in the carboxy-terminal sequence is responsible for the difference in the activity of chicken and rat liver fructose-2,6-bisphosphatase.
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/11209754
journal: Biol Chem
volume: 381(12)
pages: 1195-202
published year: 2000
authors:
  item: Zhu Z
  item: Ling S
  item: Yang QH
  item: Li L

Involvement of the chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase sequence His444-Arg-Glu-Arg in modulation of the bisphosphatase activity by its kinase domain.
type: BibliographicResource
references: http://www.ncbi.nlm.nih.gov/pubmed/11439102
journal: Biochem J
volume: 357(Pt 2)
pages: 513-20
published year: 2001
authors:
  item: Zhu Z
  item: Ling S
  item: Yang QH
  item: Li L