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Escherichia coli K-12 substr. MG1655 Enzyme: pyruvate kinase I



Gene: pykF Accession Numbers: EG10804 (EcoCyc), b1676, ECK1672

Synonyms: type I pyruvate kinase, pyruvate kinase type F

Regulation Summary Diagram: ?

Subunit composition of pyruvate kinase I = [PykF]4
         pyruvate kinase I monomer = PykF

Summary:
Pyruvate kinase I and pyruvate kinase II differ in physical and chemical properties as well as in their kinetic behavior. Although the two enzymes are under independent genetic control, they do coexist in a wide range of nutritional and metabolic states [Malcovati82, GarridoPertierr83]. The two enzymes are not interchangeable. Both show positive cooperative effects with respect to the substrate phosphoenolpyruvate [Malcovati82]. Pyruvate kinase I has a low nucleotide specificity and the 5'-diphosphates of guanosine, inosine, uridine and cytidine can all serve as phospho acceptors [Muirhead90]. Pyruvate kinase I from E. coli, unlike pyruvate kinase II, is remarkably stable [Valentini91].

A free N-terminal amino acid can be detected in both forms of pyruvate kinase; it corresponds to methionine for type I and serine for type II [Malcovati82]. Comparison with the known primary structures shows that bacterial enzymes lack a substantial portion of the N-terminal sequence with respect to pyruvate kinases from vertebrates [Valentini91].

Regulation has been described [Bledig96]. Gene expression levels, enzyme activities, metabolite concentrations and metabolic flux have been measured in a pykF mutant strain [Al04, Siddiquee04].

pykF shows differential codon adaptation, resulting in differential translation efficiency signatures, in thermophilic microbes. It was therefore predicted to play a role in the heat shock response. A pykF deletion mutant was shown to be more sensitive than wild-type specifically to heat shock, but not other stresses [Krisko14].

PykF: "pyruvate kinase, fructose 1,6-diphosphate-activated" [Kornberg73]

Locations: cytosol, membrane

Map Position: [1,753,722 -> 1,755,134] (37.8 centisomes)
Length: 1413 bp / 470 aa

Molecular Weight of Polypeptide: 50.729 kD (from nucleotide sequence)

pI: 6.08

Isozyme Sequence Similarity [Muirhead90]:

Unification Links: ASAP:ABE-0005600 , CGSC:17620 , DIP:DIP-36221N , EchoBASE:EB0797 , EcoGene:EG10804 , EcoliWiki:b1676 , ModBase:P0AD61 , OU-Microarray:b1676 , PortEco:pykF , PR:PRO_000023656 , Pride:P0AD61 , Protein Model Portal:P0AD61 , RefSeq:NP_416191 , RegulonDB:EG10804 , SMR:P0AD61 , String:511145.b1676 , Swiss-Model:P0AD61 , UniProt:P0AD61

Relationship Links: InterPro:IN-FAMILY:IPR001697 , InterPro:IN-FAMILY:IPR011037 , InterPro:IN-FAMILY:IPR015793 , InterPro:IN-FAMILY:IPR015794 , InterPro:IN-FAMILY:IPR015795 , InterPro:IN-FAMILY:IPR015806 , InterPro:IN-FAMILY:IPR015813 , InterPro:IN-FAMILY:IPR018209 , Panther:IN-FAMILY:PTHR11817 , PDB:Structure:1E0T , PDB:Structure:1E0U , PDB:Structure:1PKY , Pfam:IN-FAMILY:PF00224 , Pfam:IN-FAMILY:PF02887 , Prints:IN-FAMILY:PR01050 , Prosite:IN-FAMILY:PS00110

In Paralogous Gene Group: 354 (3 members)

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0009408 - response to heat Inferred from experiment [Krisko14]
GO:0006096 - glycolytic process Inferred by computational analysis [UniProtGOA12, UniProtGOA11, GOA01]
GO:0008152 - metabolic process Inferred by computational analysis [UniProtGOA11]
GO:0016310 - phosphorylation Inferred by computational analysis [UniProtGOA11]
Molecular Function: GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0000287 - magnesium ion binding Inferred by computational analysis [GOA01]
GO:0003824 - catalytic activity Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0004743 - pyruvate kinase activity Inferred by computational analysis [GOA01a, GOA01]
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:0030955 - potassium ion binding 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, Lasserre06]
GO:0016020 - membrane Inferred from experiment [Lasserre06]

MultiFun Terms: metabolism energy metabolism, carbon fermentation
metabolism energy metabolism, carbon glycolysis

Essentiality data for pykF knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Enzymatic reaction of: pyruvate kinase

Synonyms: pyruvate kinase, phosphoenolpyruvate kinase, phosphoenol transphosphorylase, pyruvate 2-0-phosphotransferase

EC Number: 2.7.1.40

pyruvate + ATP <=> ADP + phosphoenolpyruvate + 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.

This reaction is reversible.

In Pathways: superpathway of hexitol degradation (bacteria) , superpathway of glycolysis and Entner-Doudoroff , superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass , mixed acid fermentation , glycolysis II (from fructose-6P) , glycolysis I (from glucose-6P)

Summary:
Pyruvate kinase I is strongly activated by fructose 1,6 bisphosphate and shows sigmoid kinetics with respect to the substrate phophoenolpyruvate. The dependences of the reaction rate on the concentrations of phosphoenolpyruvate, Mg+2, and Mn+2 are sigmoid. These dependences become hyperbolic on addition of fructose 1,6 bisphosphate. The dependences of the reaction rate on the concentration of ADP are always hyperbolic. The catalytic active complex of E. coli pyruvate kinase is formed by only one conformational state of the enzyme, state R1. This state binds randomly the Mg+2 free ligands ADP or phosphoenolpyruvate. Thereafter, the active enzyme complex is formed by binding Mg+2-PEP or Mg+2ADP respectively. State R2 of coli pyruvate kinase binds randomly ADP and Mg+2. It is the only state in which Mg+2 can bind to the free enzyme. Mg+2 is not necesary for the binding of ADP, PEP an ATP. Mg+2 can inhibit either by binding to the state R2 or by lowering the concentration of Mg+2, free ADP and PEP through complex formation. ATP inhibits by virtue of three effects: increase in ionic strength, complex-formation with Mg+2 when this metal ion acts as an activator and binding to the state R3. However when conditions are such that Mg+2 is an inhibitor, then ATP is expected to activate because of complex formation with Mg+2. In general, one can say that the ligands Mg+2 and ATP cannot be classified as inhibitors or activators because their regulatory effect on the reaction rate depends on the concentration of the other ligands.[Markus80] By addition of fructose-1,6-bisphosphate the sigmoid kinetics with respect to phosphoenolpyruvate and Mg+2 is abolished and the activity of the enzyme is described by classical saturation kinetics. This is explained by exclusive binding of fructose 1,6 bisphosphate at an allosteric site of the conformational state that forms the active complex. Ca+2 is an activator of the enzyme at low Mg+2 and Ca+2 concentrations; otherwise it is an inhibitor. These effects can be understood by assuming that Ca+2 has the same binding properties as Mg+2, although it does not allow a catalytic turnover.[Boiteux83] The enzyme requires both bivalent and monovalent cations. [Muirhead90]

Cofactors or Prosthetic Groups [Comment 5]: K+ , Mg2+

Alternative Cofactors for Mg2+ [Boiteux83 ]: Mn2+

Activators (Allosteric): fructose 1,6-bisphosphate

Activators (Unknown Mechanism): Ca2+ [Comment 6]

Inhibitors (Unknown Mechanism): ATP [Comment 7] , succinyl-CoA [Comment 7] , Ca2+ [Comment 7]

Primary Physiological Regulators of Enzyme Activity: fructose 1,6-bisphosphate


Sequence Features

Feature Class Location Citations Comment
Amino-Acid-Sites-That-Bind 32
[UniProt10a]
UniProt: Substrate; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 34
[UniProt10a]
UniProt: Potassium; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 36
[UniProt10a]
UniProt: Potassium; Non-Experimental Qualifier: by similarity;
Sequence-Conflict 44
[Valentini91, UniProt10]
Alternate sequence: G → S; UniProt: (in Ref. 6; AA sequence);
Metal-Binding-Site 66
[UniProt10a]
UniProt: Potassium; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 67
[UniProt10a]
UniProt: Potassium; Non-Experimental Qualifier: by similarity;
Acetylation-Modification 76
[Zhang09, UniProt11]
UniProt: N6-acetyllysine.
Amino-Acid-Site 220
[UniProt10a]
UniProt: Transition state stabilizer; Sequence Annotation Type: site; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 222
[UniProt10a]
UniProt: Magnesium; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 245
[UniProt10a]
UniProt: Substrate; via amide nitrogen; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 246
[UniProt10a]
UniProt: Magnesium; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 278
[UniProt10a]
UniProt: Substrate; Non-Experimental Qualifier: by similarity;
Acetylation-Modification 319
[Zhang09, UniProt11]
UniProt: N6-acetyllysine.
Sequence-Conflict 379
[Speranza89, UniProt10]
Alternate sequence: Q → N; UniProt: (in Ref. 8; AA sequence);
Sequence-Conflict 401
[Speranza89, UniProt10]
Alternate sequence: T → D; UniProt: (in Ref. 8; AA sequence);
Sequence-Conflict 451 -> 470
[Ohara89, Aiba96, UniProt10]
Alternate sequence: MVSGALVPSGTTNTASVHVL → YGFWCTGTERHY; UniProt: (in Ref. 1 and 3);


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
10/20/97 Gene b1676 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10804; confirmed by SwissProt match.


References

Aiba96: Aiba H, Baba T, Hayashi K, Inada T, Isono K, Itoh T, Kasai H, Kashimoto K, Kimura S, Kitakawa M, Kitagawa M, Makino K, Miki T, Mizobuchi K, Mori H, Mori T, Motomura K, Nakade S, Nakamura Y, Nashimoto H, Nishio Y, Oshima T, Saito N, Sampei G, Horiuchi T (1996). "A 570-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 28.0-40.1 min region on the linkage map." DNA Res 3(6);363-77. PMID: 9097039

Al04: Al Zaid Siddiquee K, Arauzo-Bravo MJ, Shimizu K (2004). "Metabolic flux analysis of pykF gene knockout Escherichia coli based on 13C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations." Appl Microbiol Biotechnol 63(4);407-17. PMID: 12802531

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Bledig96: Bledig SA, Ramseier TM, Saier MH (1996). "Frur mediates catabolite activation of pyruvate kinase (pykF) gene expression in Escherichia coli." J Bacteriol 178(1);280-3. PMID: 8550429

Boiteux83: Boiteux A, Markus M, Plesser T, Hess B, Malcovati M (1983). "Analysis of progress curves. Interaction of pyruvate kinase from Escherichia coli with fructose 1,6-bisphosphate and calcium ions." Biochem J 1983;211(3);631-40. PMID: 6349612

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

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

GarridoPertierr83: Garrido-Pertierra A, Cooper RA (1983). "Evidence for two distinct pyruvate kinase genes in Escherichia coli K-12." FEBS Lett 1983;162(2);420-2. PMID: 6354749

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

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.

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

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Kornberg73: Kornberg HL, Malcovati M (1973). "Control in situ of the pyruvate kinase activity of Escherichia coli." FEBS Lett 32(2);257-9. PMID: 4582155

Krisko14: Krisko A, Copi T, Gabaldon T, Lehner B, Supek F (2014). "Inferring gene function from evolutionary change in signatures of translation efficiency." Genome Biol 15(3);R44. PMID: 24580753

Lasserre06: Lasserre JP, Beyne E, Pyndiah S, Lapaillerie D, Claverol S, Bonneu M (2006). "A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis." Electrophoresis 27(16);3306-21. PMID: 16858726

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

Malcovati82: Malcovati M, Valentini G (1982). "AMP- and fructose 1,6-bisphosphate-activated pyruvate kinases from Escherichia coli." Methods Enzymol 1982;90 Pt E;170-9. PMID: 6759852

Markus80: Markus M, Plesser T, Boiteux A, Hess B, Malcovati M (1980). "Analysis of progress curves. Rate law of pyruvate kinase type I from Escherichia coli." Biochem J 1980;189(3);421-33. PMID: 7011316

Muirhead90: Muirhead H (1990). "Isoenzymes of pyruvate kinase." Biochem Soc Trans 1990;18(2);193-6. PMID: 2379684

Ohara89: Ohara O, Dorit RL, Gilbert W (1989). "Direct genomic sequencing of bacterial DNA: the pyruvate kinase I gene of Escherichia coli." Proc Natl Acad Sci U S A 86(18);6883-7. PMID: 2674937

Siddiquee04: Siddiquee KA, Arauzo-Bravo MJ, Shimizu K (2004). "Effect of a pyruvate kinase (pykF-gene) knockout mutation on the control of gene expression and metabolic fluxes in Escherichia coli." FEMS Microbiol Lett 235(1);25-33. PMID: 15158258

Speranza89: Speranza ML, Valentini G, Iadarola P, Stoppini M, Malcovati M, Ferri G (1989). "Primary structure of three peptides at the catalytic and allosteric sites of the fructose-1,6-bisphosphate-activated pyruvate kinase from Escherichia coli." Biol Chem Hoppe Seyler 1989;370(3);211-6. PMID: 2653362

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

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 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."

Valentini91: Valentini G, Stoppini M, Speranza ML, Malcovati M, Ferri G (1991). "Bacterial pyruvate kinases have a shorter N-terminal domain." Biol Chem Hoppe Seyler 1991;372(2);91-3. PMID: 1859631

Zhang09: Zhang J, Sprung R, Pei J, Tan X, Kim S, Zhu H, Liu CF, Grishin NV, Zhao Y (2009). "Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli." Mol Cell Proteomics 8(2);215-25. PMID: 18723842

Other References Related to Gene Regulation

Kumar11: Kumar R, Shimizu K (2011). "Transcriptional regulation of main metabolic pathways of cyoA, cydB, fnr, and fur gene knockout Escherichia coli in C-limited and N-limited aerobic continuous cultures." Microb Cell Fact 10;3. PMID: 21272324

MendozaVargas09: Mendoza-Vargas A, Olvera L, Olvera M, Grande R, Vega-Alvarado L, Taboada B, Jimenez-Jacinto V, Salgado H, Juarez K, Contreras-Moreira B, Huerta AM, Collado-Vides J, Morett E (2009). "Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli." PLoS One 4(10);e7526. PMID: 19838305

Olvera09: Olvera L, Mendoza-Vargas A, Flores N, Olvera M, Sigala JC, Gosset G, Morett E, Bolivar F (2009). "Transcription analysis of central metabolism genes in Escherichia coli. Possible roles of sigma38 in their expression, as a response to carbon limitation." PLoS One 4(10);e7466. PMID: 19838295

Ponce95: Ponce E, Flores N, Martinez A, Valle F, Bolivar F (1995). "Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli: the relative roles of these enzymes in pyruvate biosynthesis." J Bacteriol 177(19);5719-22. PMID: 7559366


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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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