DB code: S00365

RLCP classification 1.15.30200.81 : Hydrolysis
CATH domain 3.40.50.1240 : Rossmann fold Catalytic domain
E.C. 3.1.3.46
CSA 2bif
M-CSA 2bif
MACiE

CATH domain Related DB codes (homologues)
3.40.50.1240 : Rossmann fold S00363 S00366 D00460 D00514

Uniprot Enzyme Name
UniprotKB Protein name Synonyms Includes RefSeq Pfam
P07953 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1
6PF-2-K/Fru-2,6-P2ASE liver isozyme
6-phosphofructo-2-kinase
EC 2.7.1.105
Fructose-2,6-bisphosphatase
EC 3.1.3.46
NP_036753.4 (Protein)
NM_012621.4 (DNA/RNA sequence)
PF01591 (6PF2K)
PF00300 (His_Phos_1)
[Graphical View]
Q9JJH5 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2
PFK-2/FBPase-2
6PF-2-K/Fru-2,6-P2ASE heart-type isozyme
RH2K
6-phosphofructo-2-kinase
EC 2.7.1.105
Fructose-2,6-bisphosphatase
EC 3.1.3.46
NP_001029136.1 (Protein)
NM_001033964.1 (DNA/RNA sequence)
NP_001029137.1 (Protein)
NM_001033965.1 (DNA/RNA sequence)
NP_536725.2 (Protein)
NM_080477.3 (DNA/RNA sequence)
PF01591 (6PF2K)
PF00300 (His_Phos_1)
[Graphical View]
O35552 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3
6PF-2-K/Fru-2,6-P2ASE brain-type isozyme
RB2K
6-phosphofructo-2-kinase
EC 2.7.1.105
Fructose-2,6-bisphosphatase
EC 3.1.3.46
NP_476476.1 (Protein)
NM_057135.1 (DNA/RNA sequence)
PF01591 (6PF2K)
PF00300 (His_Phos_1)
[Graphical View]
P25114 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4
6PF-2-K/Fru-2,6-P2ASE testis-type isozyme
6-phosphofructo-2-kinase
EC 2.7.1.105
Fructose-2,6-bisphosphatase
EC 3.1.3.46
NP_062206.1 (Protein)
NM_019333.1 (DNA/RNA sequence)
PF01591 (6PF2K)
PF00300 (His_Phos_1)
[Graphical View]

KEGG enzyme name
fructose-2,6-bisphosphate 2-phosphatase
fructose-2,6-bisphosphatase
D-fructose-2,6-bisphosphate 2-phosphohydrolase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P07953 F261_RAT Beta-D-fructose 2,6-bisphosphate + H(2)O = D- fructose 6-phosphate + phosphate. ATP + D-fructose 6-phosphate = ADP + beta-D- fructose 2,6-bisphosphate. Homodimer.
Q9JJH5 F262_RAT Beta-D-fructose 2,6-bisphosphate + H(2)O = D- fructose 6-phosphate + phosphate. ATP + D-fructose 6-phosphate = ADP + beta-D- fructose 2,6-bisphosphate. Homodimer (By similarity).
O35552 F263_RAT Beta-D-fructose 2,6-bisphosphate + H(2)O = D- fructose 6-phosphate + phosphate. ATP + D-fructose 6-phosphate = ADP + beta-D- fructose 2,6-bisphosphate.
P25114 F264_RAT Beta-D-fructose 2,6-bisphosphate + H(2)O = D- fructose 6-phosphate + phosphate. ATP + D-fructose 6-phosphate = ADP + beta-D- fructose 2,6-bisphosphate. Homodimer.

KEGG Pathways
Map code Pathways E.C.
MAP00051 Fructose and mannose metabolism

Compound table
Substrates Products Intermediates
KEGG-id C00665 C00001 C00085 C00009
E.C.
Compound D-Fructose 2,6-bisphosphate H2O D-Fructose 6-phosphate Orthophosphate
Type carbohydrate,phosphate group/phosphate ion H2O carbohydrate,phosphate group/phosphate ion phosphate group/phosphate ion
ChEBI 28602
15377
61553
26078
PubChem 105021
22247451
962
439160
1004
22486802
1bifA02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Analogue:PO4 Bound:PO4 Unbound
1fbtA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Bound:PO4 Unbound
1fbtB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Bound:PO4 Unbound
1tipA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:F6P Unbound Bound:H2P (Phosphohistidine intermediate)
1tipB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:F6P Unbound Bound:H2P (Phosphohistidine intermediate)
2bifA02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:F6P Bound:PO4 Unbound
2bifB02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:F6P Bound:PO4 Unbound
3bifA02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Analogue:PO4 Bound:PO4 Unbound

Reference for Active-site residues
resource references E.C.
Swiss-prot;HIS 8 forms phosphohistidine intermediate.

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1bifA02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 325;HIS 390 HIS 256
1fbtA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 77;HIS 142 HIS 8
1fbtB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 77;HIS 142 HIS 8
1tipA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 78;HIS 143 NEP 9 NEP (phosphorylated HIS)
1tipB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 78;HIS 143 NEP 9 NEP (phosphorylated HIS)
2bifA02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 325;HIS 390 mutant H256A
2bifB02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 325;HIS 390 mutant H256A
3bifA02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 325;HIS 390 HIS 256

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[6]
Fig.4, p.6560-6561 3
[8]
Fig.5, p.6015-6016 2
[9]
Fig.5, p.1022-1024 2
[10]
Fig.3, p.616-617 2
[12]
p.4478
[13]
Fig.7, p.2175 2
[14]
Fig.5, p.2181-2183 3
[15]
Fig.6, p.9759-9761 5

References
[1]
Resource
Comments
Medline ID
PubMed ID 6319392
Journal J Biol Chem
Year 1984
Volume 259(2)
Pages 949-58
Authors Pilkis SJ, Regen DM, Stewart HB, Pilkis J, Pate TM, El-Maghrabi MR
Title Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID 3013863
Journal J Biol Chem
Year 1986
Volume 261(19)
Pages 8793-8
Authors Stewart HB, el-Maghrabi MR, Pilkis SJ
Title Mechanism of activation of fructose-2,6-bisphosphatase by cAMP-dependent protein kinase.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID 2539378
Journal J Biol Chem
Year 1989
Volume 264(11)
Pages 6344-8
Authors Kitamura K, Uyeda K, Hartman FC, Kangawa K, Matsuo H
Title Catalytic site of rat liver and bovine heart fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Identification of fructose 6-phosphate binding site.
Related PDB
Related UniProtKB P07953
[4]
Resource
Comments
Medline ID
PubMed ID 2557623
Journal Proc Natl Acad Sci U S A
Year 1989
Volume 86(24)
Pages 9642-6
Authors Bazan JF, Fletterick RJ, Pilkis SJ
Title Evolution of a bifunctional enzyme: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID 1339450
Journal J Biol Chem
Year 1992
Volume 267(7)
Pages 4416-23
Authors Kurland IJ, el-Maghrabi MR, Correia JJ, Pilkis SJ
Title 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.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID 1313012
Journal J Biol Chem
Year 1992
Volume 267(10)
Pages 6556-62
Authors Lin K, Li L, Correia JJ, Pilkis SJ
Title Glu327 is part of a catalytic triad in rat liver fructose-2,6-bisphosphatase.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID 7755565
Journal Biochem J
Year 1995
Volume 308 (Pt 1)
Pages 189-95
Authors Okar DA, Kakalis LT, Narula SS, Armitage IM, Pilkis SJ
Title Identification of transient intermediates in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by 31P-NMR spectroscopy.
Related PDB
Related UniProtKB
[8]
Resource
Comments X-ray crystallography (2.5 Angstroms)
Medline ID
PubMed ID 8634242
Journal Biochemistry
Year 1996
Volume 35(19)
Pages 6010-6019
Authors Lee YH, Ogata C, Pflugrath JW, Levitt DG, Sarma R, Banaszak LJ, Pilkis SJ
Title Crystal structure of the rat liver fructose-2,6-bisphosphatase based on selenomethionine multiwavelength anomalous dispersion phases.
Related PDB 1fbt
Related UniProtKB
[9]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS)
Medline ID
PubMed ID 8805587
Journal Structure
Year 1996
Volume 4(9)
Pages 1017-29
Authors Hasemann CA, Istvan ES, Uyeda K, Deisenhofer J
Title The crystal structure of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase reveals distinct domain homologies.
Related PDB 1bif 3bif
Related UniProtKB P25114
[10]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 9253407
Journal Nat Struct Biol
Year 1997
Volume 4(8)
Pages 615-618
Authors Lee YH, Olson TW, Ogata CM, Levitt DG, Banaszak LJ, Lange AJ
Title Crystal structure of a trapped phosphoenzyme during a catalytic reaction.
Related PDB 1tip
Related UniProtKB P07953
[11]
Resource
Comments
Medline ID
PubMed ID 9760241
Journal Biochemistry
Year 1998
Volume 37(40)
Pages 14057-64
Authors Helms MK, Hazlett TL, Mizuguchi H, Hasemann CA, Uyeda K, Jameson DM
Title Site-directed mutants of rat testis fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase: localization of conformational alterations induced by ligand binding.
Related PDB
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID 10194369
Journal Biochemistry
Year 1999
Volume 38(14)
Pages 4471-9
Authors Okar DA, Live DH, Kirby TL, Karschnia EJ, von Weymarn LB, Armitage IM, 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.
Related PDB
Related UniProtKB
[13]
Resource
Comments
Medline ID
PubMed ID 9890979
Journal J Biol Chem
Year 1999
Volume 274(4)
Pages 2166-75
Authors Mizuguchi H, Cook PF, Tai CH, Hasemann CA, Uyeda K
Title Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway.
Related PDB
Related UniProtKB
[14]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.4 ANGSTROMS) OF MUTANT ALA-256
Medline ID
PubMed ID 9890980
Journal J Biol Chem
Year 1999
Volume 274(4)
Pages 2176-84
Authors Yuen MH, Mizuguchi H, Lee YH, Cook PF, Uyeda K, 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.
Related PDB 2bif
Related UniProtKB P25114
[15]
Resource
Comments
Medline ID
PubMed ID 10933792
Journal Biochemistry
Year 2000
Volume 39(32)
Pages 9754-62
Authors Okar DA, Live DH, Devany MH, Lange AJ
Title Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy.
Related PDB
Related UniProtKB
[16]
Resource
Comments
Medline ID
PubMed ID 11209754
Journal Biol Chem
Year 2000
Volume 381(12)
Pages 1195-202
Authors Zhu Z, Ling S, Yang QH, Li L
Title The difference in the carboxy-terminal sequence is responsible for the difference in the activity of chicken and rat liver fructose-2,6-bisphosphatase.
Related PDB
Related UniProtKB
[17]
Resource
Comments
Medline ID
PubMed ID 11439102
Journal Biochem J
Year 2001
Volume 357(Pt 2)
Pages 513-20
Authors Zhu Z, Ling S, Yang QH, Li L
Title 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.
Related PDB
Related UniProtKB

Comments
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.

Created Updated
2002-07-09 2009-02-26