EzCatDB: S00365

DB codeS00365
RLCP classification1.15.30200.81
CATH domainDomain 13.40.50.1240Catalytic domain
E.C.3.1.3.46
CSA2bif

CATH domainRelated DB codes (homologues)
3.40.50.1240S00363,S00366,D00460,D00514

Enzyme Name
Swiss-protKEGG

P07953Q9JJH5O35552P25114
Protein name6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 16-phosphofructo-2-kinase/fructose-2,6-biphosphatase 26-phosphofructo-2-kinase/fructose-2,6-biphosphatase 36-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4fructose-2,6-bisphosphate 2-phosphatase
fructose-2,6-bisphosphatase
D-fructose-2,6-bisphosphate 2-phosphohydrolase
Synonyms6PF-2-K/Fru-2,6-P2ASE liver isozyme
PFK-2/FBPase-2
6PF-2-K/Fru-2,6-P2ASE heart-type isozyme
RH2K
6PF-2-K/Fru-2,6-P2ASE brain-type isozyme
RB2K
6PF-2-K/Fru-2,6-P2ASE testis-type isozyme
Includes6-phosphofructo-2-kinase
   EC 2.7.1.105
Fructose-2,6-bisphosphatase
   EC 3.1.3.46
6-phosphofructo-2-kinase
   EC 2.7.1.105
Fructose-2,6-bisphosphatase
   EC 3.1.3.46
6-phosphofructo-2-kinase
   EC 2.7.1.105
Fructose-2,6-bisphosphatase
   EC 3.1.3.46
6-phosphofructo-2-kinase
   EC 2.7.1.105
Fructose-2,6-bisphosphatase
   EC 3.1.3.46

KEGG pathways
MAP codePathways
MAP00051Fructose and mannose metabolism

Swiss-prot:Accession NumberP07953Q9JJH5O35552P25114
Entry nameF261_RATF262_RATF263_RATF264_RAT
ActivityBeta-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.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.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.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.
SubunitHomodimer.Homodimer (By similarity).
Homodimer.
Subcellular location



Cofactor





SubstratesProductsintermediates
KEGG-idC00665C00001C00085C00009
CompoundD-Fructose 2,6-bisphosphateH2OD-Fructose 6-phosphateOrthophosphate
Typecarbohydrate,phosphate group/phosphate ionH2Ocarbohydrate,phosphate group/phosphate ionphosphate group/phosphate ion
1bifA02Unbound
Analogue:PO4Bound:PO4Unbound
1fbtAUnbound
UnboundBound:PO4Unbound
1fbtBUnbound
UnboundBound:PO4Unbound
1tipAUnbound
Bound:F6PUnboundBound:H2P(Phosphohistidine intermediate)
1tipBUnbound
Bound:F6PUnboundBound:H2P(Phosphohistidine intermediate)
2bifA02Unbound
Bound:F6PBound:PO4Unbound
2bifB02Unbound
Bound:F6PBound:PO4Unbound
3bifA02Unbound
Analogue:PO4Bound:PO4Unbound

Active-site residues
resource
Swiss-prot;HIS 8 forms phosphohistidine intermediate.
pdbCatalytic residuesModified residuescomment
1bifA02GLU 325;HIS 390
HIS 256

1fbtAGLU  77;HIS 142
HIS   8

1fbtBGLU  77;HIS 142
HIS   8

1tipAGLU  78;HIS 143
NEP   9
NEP (phosphorylated HIS)
1tipBGLU  78;HIS 143
NEP   9
NEP (phosphorylated HIS)
2bifA02GLU 325;HIS 390
       
mutant H256A
2bifB02GLU 325;HIS 390
       
mutant H256A
3bifA02GLU 325;HIS 390
HIS 256


References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[6]Fig.4, p.6560-65613
[8]Fig.5, p.6015-60162
[9]Fig.5, p.1022-10242
[10]Fig.3, p.616-6172
[12]p.4478
[13]Fig.7, p.21752
[14]Fig.5, p.2181-21833
[15]Fig.6, p.9759-97615

references
[1]
PubMed ID6319392
JournalJ Biol Chem
Year1984
Volume259(2)
Pages949-58
AuthorsPilkis SJ, Regen DM, Stewart HB, Pilkis J, Pate TM, El-Maghrabi MR
TitleEvidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation.
[2]
PubMed ID3013863
JournalJ Biol Chem
Year1986
Volume261(19)
Pages8793-8
AuthorsStewart HB, el-Maghrabi MR, Pilkis SJ
TitleMechanism of activation of fructose-2,6-bisphosphatase by cAMP-dependent protein kinase.
[3]
PubMed ID2539378
JournalJ Biol Chem
Year1989
Volume264(11)
Pages6344-8
AuthorsKitamura K, Uyeda K, Hartman FC, Kangawa K, Matsuo H
TitleCatalytic site of rat liver and bovine heart fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Identification of fructose 6-phosphate binding site.
Related Swiss-protP07953
[4]
PubMed ID2557623
JournalProc Natl Acad Sci U S A
Year1989
Volume86(24)
Pages9642-6
AuthorsBazan JF, Fletterick RJ, Pilkis SJ
TitleEvolution of a bifunctional enzyme: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
[5]
PubMed ID1339450
JournalJ Biol Chem
Year1992
Volume267(7)
Pages4416-23
AuthorsKurland IJ, el-Maghrabi MR, Correia JJ, Pilkis SJ
TitleRat 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.
[6]
PubMed ID1313012
JournalJ Biol Chem
Year1992
Volume267(10)
Pages6556-62
AuthorsLin K, Li L, Correia JJ, Pilkis SJ
TitleGlu327 is part of a catalytic triad in rat liver fructose-2,6-bisphosphatase.
[7]
PubMed ID7755565
JournalBiochem J
Year1995
Volume308 (Pt 1)
Pages189-95
AuthorsOkar DA, Kakalis LT, Narula SS, Armitage IM, Pilkis SJ
TitleIdentification of transient intermediates in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by 31P-NMR spectroscopy.
[8]
CommentsX-ray crystallography (2.5 Angstroms)
PubMed ID8634242
JournalBiochemistry
Year1996
Volume35(19)
Pages6010-6019
AuthorsLee YH, Ogata C, Pflugrath JW, Levitt DG, Sarma R, Banaszak LJ, Pilkis SJ
TitleCrystal structure of the rat liver fructose-2,6-bisphosphatase based on selenomethionine multiwavelength anomalous dispersion phases.
Related PDB1fbt
[9]
CommentsX-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS)
PubMed ID8805587
JournalStructure
Year1996
Volume4(9)
Pages1017-29
AuthorsHasemann CA, Istvan ES, Uyeda K, Deisenhofer J
TitleThe crystal structure of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase reveals distinct domain homologies.
Related PDB1bif,3bif
Related Swiss-protP25114
[10]
CommentsX-ray crystallography
PubMed ID9253407
JournalNat Struct Biol
Year1997
Volume4(8)
Pages615-618
AuthorsLee YH, Olson TW, Ogata CM, Levitt DG, Banaszak LJ, Lange AJ
TitleCrystal structure of a trapped phosphoenzyme during a catalytic reaction.
Related PDB1tip
Related Swiss-protP07953
[11]
PubMed ID9760241
JournalBiochemistry
Year1998
Volume37(40)
Pages14057-64
AuthorsHelms MK, Hazlett TL, Mizuguchi H, Hasemann CA, Uyeda K, Jameson DM
TitleSite-directed mutants of rat testis fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase: localization of conformational alterations induced by ligand binding.
[12]
PubMed ID10194369
JournalBiochemistry
Year1999
Volume38(14)
Pages4471-9
AuthorsOkar DA, Live DH, Kirby TL, Karschnia EJ, von Weymarn LB, Armitage IM, Lange AJ
TitleThe 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.
[13]
PubMed ID9890979
JournalJ Biol Chem
Year1999
Volume274(4)
Pages2166-75
AuthorsMizuguchi H, Cook PF, Tai CH, Hasemann CA, Uyeda K
TitleReaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway.
[14]
CommentsX-RAY CRYSTALLOGRAPHY (2.4 ANGSTROMS) OF MUTANT ALA-256
PubMed ID9890980
JournalJ Biol Chem
Year1999
Volume274(4)
Pages2176-84
AuthorsYuen MH, Mizuguchi H, Lee YH, Cook PF, Uyeda K, Hasemann CA
TitleCrystal 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 PDB2bif
Related Swiss-protP25114
[15]
PubMed ID10933792
JournalBiochemistry
Year2000
Volume39(32)
Pages9754-62
AuthorsOkar DA, Live DH, Devany MH, Lange AJ
TitleMechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy.
[16]
PubMed ID11209754
JournalBiol Chem
Year2000
Volume381(12)
Pages1195-202
AuthorsZhu Z, Ling S, Yang QH, Li L
TitleThe difference in the carboxy-terminal sequence is responsible for the difference in the activity of chicken and rat liver fructose-2,6-bisphosphatase.
[17]
PubMed ID11439102
JournalBiochem J
Year2001
Volume357(Pt 2)
Pages513-20
AuthorsZhu Z, Ling S, Yang QH, Li L
TitleInvolvement 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.

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.

createdupdated
2002-07-092009-02-26


Copyright: Nozomi Nagano, JST & CBRC-AIST
Funded by PRESTO/Japan Science and Technology Corporation (JST) (December 2001 - November 2004)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2006)
Funded by Grant-in-Aid for Scientific Research (B)/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (September 2005 - September 2010)
Funded by BIRD/Japan Science and Technology Corporation (JST) (October 2007 - September 2010)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2011 - March 2012)

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