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MetaCyc Enzyme: 6-phosphofructokinase II

Gene: pfkB Accession Numbers: EG10700 (MetaCyc), b1723, ECK1721

Synonyms: PFK II

Species: Escherichia coli K-12 substr. MG1655

Subunit composition of 6-phosphofructokinase II = [PfkB]2
         6-phosphofructokinase-2 monomer = PfkB

Phosphofructokinase (Pfk) catalyzes the phosphorylation of fructose-6-phosphate on the C1 carbon during glycolysis. E. coli contains two Pfk isozymes, Pfk-1 and Pfk-2, which do not share sequence similarity [Hellinga85]. Only less than 5% of Pfk activity in the wild-type cells can be attributed to Pfk-2 [Kotlarz75].

Pfk-2 is a member of the ribokinase family of sugar kinases. Pfk-2, unlike Pfk-1, does not show cooperative interaction with fructose-6-phosphate, inhibition by PEP or activation by ADP [Fraenkel73, Babul78]. MgATP2- is the true substrate of the enzyme [Guixe85].

Allosteric binding of MgATP2- to dimeric Pfk-2 leads to tetramerization and inhibition of enzymatic activity. Increased sugar-phosphate concentration reverts both effects [Babul78, Guixe85, Guixe88, Kotlarz81, Guixe98, Guixe00], while K+ enhances allosteric inhibition by MgATP2- [Baez13]. The kinetics of allosteric inhibition by MgATP has been determined, and the interplay of substrate binding and allosteric binding of MgATP has been analyzed [Cabrera11]. The dimeric state of Pfk-2 is essential for the stability and activity of the enzyme [Caniuguir05]. Studies on the unfolding and folding pathways of the dimeric and tetrameric forms of the enzyme have been performed [Baez07, Baez09, Baez11, Baez12, RamirezSarmient13]. A catalytic Mg2+ ion in addition to the one present in the MgATP2- complex is required for activity of Pfk-2 [Parducci06, RivasPardo11].

The conserved Glu190 residue is important for binding of Mg2+ and contributes to the mechanism of phosphate inhibition [Parducci06]. An N-terminal proteolytic fragment of Pfk-2 is a dimer and retains the ability to bind fructose-6-phosphase and MgATP2-, but is catalytically inactive [Cabrera02]. The Cys295 residue affects the active site properties and may be involved in dimer-dimer interactions [Caniuguir05]. R90 appears to participate in binding of the phosphorylated substrate, and D256 is involved in phosphoryl transfer [Cabrera10]. Multiple sequence alignments have identified phylogenetically conserved active site residues. Y306 is required for quarternary packing involved in the dimer-tetramer conversion, while L307 is required for the catalytic MgATP2- binding site [Baez08].

A crystal structure of Pfk-2 in the tetrameric form inhibited by MgATP has been solved at 1.98 Å resolution [Cabrera06, Cabrera08]. Comparison of this structure with a crystal structure of Pfk-2 in complex with fructose-6-phosphate suggests negative interplay between fructose-6-phosphate binding and MgATP binding [Cabrera11].

In strains with active Pfk-1, pfkB may be deleted without apparent effect. However, in the absence of Pfk-1, slow growth on sugars depends on the presence of Pfk-2. Its loss causes complete inability to grow [Vinopal75, Daldal81, Daldal83].

Pfk-2 can also use tagatose-6-phosphate as a substrate [Babul78]. This reaction is part of the galactitol catabolism pathway [Neidhardt96].

The pfkB gene utilizes rare codons and has a poor consensus promoter sequence, common attributes of weakly expressed genes [Daldal84]. Transcription of pfkB is increased by growth on glycerol as the carbon source compared to growth on glucose [MartinezGomez12].

Citations: [Kim13a]

Locations: cytosol

Map Position: [1,804,394 -> 1,805,323]

Molecular Weight of Polypeptide: 32.456 kD (from nucleotide sequence), 36.0 kD (experimental) [Kotlarz77 ]

Molecular Weight of Multimer: 71.0 kD (experimental) [Kotlarz77]

pI: 6.05

Unification Links: ASAP:ABE-0005748 , CGSC:412 , DIP:DIP-10465N , EchoBASE:EB0694 , EcoGene:EG10700 , EcoliWiki:b1723 , Mint:MINT-8178151 , ModBase:P06999 , OU-Microarray:b1723 , PortEco:pfkB , PR:PRO_000023515 , Pride:P06999 , Protein Model Portal:P06999 , RefSeq:NP_416237 , RegulonDB:EG10700 , SMR:P06999 , String:511145.b1723 , UniProt:P06999

Relationship Links: InterPro:IN-FAMILY:IPR002173 , InterPro:IN-FAMILY:IPR011611 , InterPro:IN-FAMILY:IPR017583 , PDB:Structure:3CQD , PDB:Structure:3N1C , PDB:Structure:3UMO , PDB:Structure:3UMP , PDB:Structure:3UQD , PDB:Structure:3UQE , Pfam:IN-FAMILY:PF00294 , Prosite:IN-FAMILY:PS00583 , Prosite:IN-FAMILY:PS00584

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006096 - glycolytic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, UniProtGOA11, Vinopal75]
GO:0006974 - cellular response to DNA damage stimulus Inferred from experiment [Khil02]
GO:0046835 - carbohydrate phosphorylation Inferred by computational analysis Inferred from experiment [Babul78, GOA01a]
GO:0005975 - carbohydrate metabolic process Inferred by computational analysis [GOA01]
GO:0016310 - phosphorylation Inferred by computational analysis [UniProtGOA11]
Molecular Function: GO:0000287 - magnesium ion binding Inferred from experiment [Parducci06]
GO:0003872 - 6-phosphofructokinase activity Inferred from experiment Inferred by computational analysis [GOA01a, Babul78]
GO:0009024 - tagatose-6-phosphate kinase activity Inferred from experiment [Babul78]
GO:0042802 - identical protein binding Inferred from experiment [Baez11]
GO:0042803 - protein homodimerization activity Inferred from experiment [Kotlarz77]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11]
GO:0016301 - kinase activity Inferred by computational analysis [UniProtGOA11]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
GO:0016773 - phosphotransferase activity, alcohol group as acceptor Inferred by computational analysis [GOA01]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05]

MultiFun Terms: metabolism carbon utilization carbon compounds

Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Enzymatic reaction of: 6-phosphofructokinase

Synonyms: fructose-6-p-1-kinase, fructose-6-phosphate-1-phosphotransferase

EC Number:

ATP + β-D-fructofuranose 6-phosphate <=> ADP + fructose 1,6-bisphosphate + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

In Pathways: superpathway of hexitol degradation (bacteria) , superpathway of N-acetylneuraminate degradation , superpathway of glycolysis and Entner-Doudoroff , superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass , 1,3-propanediol biosynthesis (engineered) , glycolysis II (from fructose 6-phosphate) , glycolysis I (from glucose 6-phosphate)

Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

The reverse reaction proceeds at ~1% of the rate of the forward reaction and is sensitive to inhibition by ATP [Babul78].

Mn2+ can substitute for Mg2+ as the divalent metal cofactor [RivasPardo11].

Cofactors or Prosthetic Groups: Mg2+ [RivasPardo11, Parducci06]

Inhibitors (Allosteric): ATP [Guixe98, Kotlarz81]

Inhibitors (Competitive): fructose 1,6-bisphosphate [Campos84, Babul78]

Inhibitors (Unknown Mechanism): K+ [Baez13] , phosphate [Parducci06]

Primary Physiological Regulators of Enzyme Activity: ATP

Kinetic Parameters:

Km (μM)
β-D-fructofuranose 6-phosphate
fructose 1,6-bisphosphate

Enzymatic reaction of: tagatose-6-phosphate kinase (6-phosphofructokinase II)

EC Number:

D-tagatofuranose 6-phosphate + ATP <=> D-tagatofuranose 1,6-bisphosphate + ADP + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

In Pathways: superpathway of hexitol degradation (bacteria) , galactitol degradation

Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Kinetic Parameters:

Km (μM)
D-tagatofuranose 6-phosphate

Sequence Features

Feature Class Location Citations Comment
Sequence-Conflict 26 -> 38
[Daldal83, Daldal84, UniProt10]
Alternate sequence: ENCAVPHRCSNP; UniProt: (in Ref. 1 and 5);
Sequence-Conflict 155 -> 171
[Daldal84, UniProt10]
Alternate sequence: LRKNKGSAASSTVLGQG; UniProt: (in Ref. 1; AAA24320);
Sequence-Conflict 245 -> 246
[Daldal84, UniProt10]
Alternate sequence: AL; UniProt: (in Ref. 1; AAA24321);
Sequence-Conflict 257 -> 258
[Daldal84, UniProt10]
Alternate sequence: RL; UniProt: (in Ref. 1; AAA24321);

10/20/97 Gene b1723 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10700; confirmed by SwissProt match.


Babul78: Babul J (1978). "Phosphofructokinases from Escherichia coli. Purification and characterization of the nonallosteric isozyme." J Biol Chem 1978;253(12);4350-5. PMID: 149128

Baez07: Baez M, Cabrera R, Guixe V, Babul J (2007). "Unfolding pathway of the dimeric and tetrameric forms of phosphofructokinase-2 from Escherichia coli." Biochemistry 46(20);6141-8. PMID: 17469854

Baez08: Baez M, Merino F, Astorga G, Babul J (2008). "Uncoupling the MgATP-induced inhibition and aggregation of Escherichia coli phosphofructokinase-2 by C-terminal mutations." FEBS Lett 582(13);1907-12. PMID: 18501195

Baez09: Baez M, Babul J (2009). "Reversible unfolding of dimeric phosphofructokinase-2 from Escherichia coli reveals a dominant role of inter-subunit contacts for stability." FEBS Lett 583(12);2054-60. PMID: 19465020

Baez11: Baez M, Wilson CA, Babul J (2011). "Folding kinetic pathway of phosphofructokinase-2 from Escherichia coli: a homodimeric enzyme with a complex domain organization." FEBS Lett 585(14);2158-64. PMID: 21627967

Baez12: Baez M, Wilson CA, Ramirez-Sarmiento CA, Guixe V, Babul J (2012). "Expanded monomeric intermediate upon cold and heat unfolding of phosphofructokinase-2 from Escherichia coli." Biophys J 103(10);2187-94. PMID: 23200052

Baez13: Baez M, Cabrera R, Pereira HM, Blanco A, Villalobos P, Ramirez-Sarmiento CA, Caniuguir A, Guixe V, Garratt RC, Babul J (2013). "A ribokinase family conserved monovalent cation binding site enhances the MgATP-induced inhibition in E. coli phosphofructokinase-2." Biophys J 105(1);185-93. PMID: 23823238

Cabrera02: Cabrera R, Guixe V, Alfaro J, Rodriguez PH, Babul J (2002). "Ligand-dependent structural changes and limited proteolysis of Escherichia coli phosphofructokinase-2." Arch Biochem Biophys 406(2);289-95. PMID: 12361717

Cabrera06: Cabrera R, Caniuguir A, Ambrosio AL, Guixe V, Garratt RC, Babul J (2006). "Crystallization and preliminary crystallographic analysis of the tetrameric form of phosphofructokinase-2 from Escherichia coli, a member of the ribokinase family." Acta Crystallogr Sect F Struct Biol Cryst Commun 62(Pt 9);935-7. PMID: 16946484

Cabrera08: Cabrera R, Ambrosio AL, Garratt RC, Guixe V, Babul J (2008). "Crystallographic structure of phosphofructokinase-2 from Escherichia coli in complex with two ATP molecules. Implications for substrate inhibition." J Mol Biol 383(3);588-602. PMID: 18762190

Cabrera10: Cabrera R, Babul J, Guixe V (2010). "Ribokinase family evolution and the role of conserved residues at the active site of the PfkB subfamily representative, Pfk-2 from Escherichia coli." Arch Biochem Biophys 502(1);23-30. PMID: 20599671

Cabrera11: Cabrera R, Baez M, Pereira HM, Caniuguir A, Garratt RC, Babul J (2011). "The crystal complex of phosphofructokinase-2 of Escherichia coli with fructose-6-phosphate: kinetic and structural analysis of the allosteric ATP inhibition." J Biol Chem 286(7);5774-83. PMID: 21147773

Campos84: Campos G, Guixe V, Babul J (1984). "Kinetic mechanism of phosphofructokinase-2 from Escherichia coli. A mutant enzyme with a different mechanism." J Biol Chem 259(10);6147-52. PMID: 6233271

Caniuguir05: Caniuguir A, Cabrera R, Baez M, Vasquez CC, Babul J, Guixe V (2005). "Role of Cys-295 on subunit interactions and allosteric regulation of phosphofructokinase-2 from Escherichia coli." FEBS Lett 579(11);2313-8. PMID: 15848164

Daldal81: Daldal F, Fraenkel DG (1981). "Tn10 insertions in the pfkB region of Escherichia coli." J Bacteriol 147(3);935-43. PMID: 6268614

Daldal83: Daldal F (1983). "Molecular cloning of the gene for phosphofructokinase-2 of Escherichia coli and the nature of a mutation, pfkB1, causing a high level of the enzyme." J Mol Biol 1983;168(2);285-305. PMID: 6310120

Daldal84: Daldal F (1984). "Nucleotide sequence of gene pfkB encoding the minor phosphofructokinase of Escherichia coli K-12." Gene 1984;28(3);337-42. PMID: 6235149

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Fraenkel73: Fraenkel DG, Kotlarz D, Buc H (1973). "Two fructose 6-phosphate kinase activities in Escherichia coli." J Biol Chem 248(13);4865-6. PMID: 4268865

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

Guixe00: Guixe V (2000). "Chemical modification of SH groups of E. coli phosphofructokinase-2 induces subunit dissociation: monomers are inactive but preserve ligand binding properties." Arch Biochem Biophys 376(2);313-9. PMID: 10775417

Guixe85: Guixe V, Babul J (1985). "Effect of ATP on phosphofructokinase-2 from Escherichia coli. A mutant enzyme altered in the allosteric site for MgATP." J Biol Chem 1985;260(20);11001-5. PMID: 3161887

Guixe88: Guixe V, Babul J (1988). "Influence of ligands on the aggregation of the normal and mutant forms of phosphofructokinase 2 of Escherichia coli." Arch Biochem Biophys 1988;264(2);519-24. PMID: 2969698

Guixe98: Guixe V, Rodriguez PH, Babul J (1998). "Ligand-induced conformational transitions in Escherichia coli phosphofructokinase 2: evidence for an allosteric site for MgATP2-." Biochemistry 37(38);13269-75. PMID: 9748334

Hellinga85: Hellinga HW, Evans PR (1985). "Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase." Eur J Biochem 1985;149(2);363-73. PMID: 3158524

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Khil02: Khil PP, Camerini-Otero RD (2002). "Over 1000 genes are involved in the DNA damage response of Escherichia coli." Mol Microbiol 44(1);89-105. PMID: 11967071

Kim13a: Kim HU, Kim WJ, Lee SY (2013). "Flux-coupled genes and their use in metabolic flux analysis." Biotechnol J. PMID: 23420780

Kotlarz75: Kotlarz D, Garreau H, Buc H (1975). "Regulation of the amount and of the activity of phosphofructokinases and pyruvate kinases in Escherichia coli." Biochim Biophys Acta 381(2);257-68. PMID: 122902

Kotlarz77: Kotlarz D, Buc H (1977). "Two Escherichia coli fructose-6-phosphate kinases. Preparative purification, oligomeric structure and immunological studies." Biochim Biophys Acta 484(1);35-48. PMID: 70226

Kotlarz81: Kotlarz D, Buc H (1981). "Regulatory properties of phosphofructokinase 2 from Escherichia coli." Eur J Biochem 1981;117(3);569-74. PMID: 6456900

LopezCampistrou05: Lopez-Campistrous A, Semchuk P, Burke L, Palmer-Stone T, Brokx SJ, Broderick G, Bottorff D, Bolch S, Weiner JH, Ellison MJ (2005). "Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth." Mol Cell Proteomics 4(8);1205-9. PMID: 15911532

MartinezGomez12: Martinez-Gomez K, Flores N, Castaneda HM, Martinez-Batallar G, Hernandez-Chavez G, Ramirez OT, Gosset G, Encarnacion S, Bolivar F (2012). "New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol." Microb Cell Fact 11;46. PMID: 22513097

Neidhardt96: Neidhardt FC, Curtiss III R, Ingraham JL, Lin ECC, Low Jr KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE "Escherichia coli and Salmonella, Cellular and Molecular Biology, Second Edition." American Society for Microbiology, Washington, D.C., 1996.

Parducci06: Parducci RE, Cabrera R, Baez M, Guixe V (2006). "Evidence for a catalytic Mg2+ ion and effect of phosphate on the activity of Escherichia coli phosphofructokinase-2: regulatory properties of a ribokinase family member." Biochemistry 45(30);9291-9. PMID: 16866375

RamirezSarmient13: Ramirez-Sarmiento CA, Baez M, Wilson CA, Babul J, Komives EA, Guixe V (2013). "Observation of solvent penetration during cold denaturation of E. coli phosphofructokinase-2." Biophys J 104(10);2254-63. PMID: 23708365

RivasPardo11: Rivas-Pardo JA, Caniuguir A, Wilson CA, Babul J, Guixe V (2011). "Divalent metal cation requirements of phosphofructokinase-2 from E. coli. Evidence for a high affinity binding site for Mn2+." Arch Biochem Biophys 505(1);60-6. PMID: 20887711

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Vinopal75: Vinopal RT, Fraenkel DG (1975). "PfkB and pfkC loci of Escherichia coli." J Bacteriol 122(3);1153-61. PMID: 125264

Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 18.5 on Thu Mar 5, 2015, BIOCYC11A.