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
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 28602	15377 15377	61553 61553	26078 26078
PubChem	105021 105021	22247451 962 22247451 962	439160 439160	1004 22486802 1004 22486802

1bifA02	Unbound		Analogue:PO4	Bound:PO4	Unbound
1fbtA	Unbound		Unbound	Bound:PO4	Unbound
1fbtB	Unbound		Unbound	Bound:PO4	Unbound
1tipA	Unbound		Bound:F6P	Unbound	Bound:H2P (Phosphohistidine intermediate)
1tipB	Unbound		Bound:F6P	Unbound	Bound:H2P (Phosphohistidine intermediate)
2bifA02	Unbound		Bound:F6P	Bound:PO4	Unbound
2bifB02	Unbound		Bound:F6P	Bound:PO4	Unbound
3bifA02	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	GLU 325;HIS 390		HIS 256
1fbtA	GLU 77;HIS 142		HIS 8
1fbtB	GLU 77;HIS 142		HIS 8
1tipA	GLU 78;HIS 143		NEP 9		NEP (phosphorylated HIS)
1tipB	GLU 78;HIS 143		NEP 9		NEP (phosphorylated HIS)
2bifA02	GLU 325;HIS 390				mutant H256A
2bifB02	GLU 325;HIS 390				mutant H256A
3bifA02	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.

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