|Gene:||pykF||Accession Numbers: EG10804 (EcoCyc), b1676, ECK1672|
Synonyms: type I pyruvate kinase, pyruvate kinase type F
Pyruvate kinase is a key allosteric enzyme of glycolysis, catalyzing one of the two substrate-level phosphorylation steps that generate ATP. The second product, pyruvate, is either used in many metabolic pathways to synthesize cell materials, or is further oxidized via the TCA cycle. The transfer of the phosphoryl group of phosphoenolpyruvate to ADP to form pyruvate and ATP is the last step in the glycolytic pathway and is irreversible under physiological conditions [Valentini00, Mattevi95].
Two forms of pyruvate kinase have been described in E. coli. Pyruvate kinase I encoded by pykF and pyruvate kinase II encoded by pykA 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 [Malcovati73, Gibriel75, Valentini79, Malcovati82, GarridoPertierr83, Valentini93, Ponce95].
The two enzymes are not interchangeable. Both show positive cooperative effects with respect to their substrate phosphoenolpyruvate, although pyruvate kinase II shows only limited cooperativity. Pyruvate kinase I is activated by fructose 1,6-bisphosphate and inhibited by ATP and succinyl-CoA, whereas pyruvate kinase II is allosterically activated by AMP and several sugar phosphates. Both enzymes are homotetramers [Waygood75, Mort78, Somani77, Valentini79, Malcovati82, Valentini91].
Pyruvate kinase I has been purified and characterized from cell extracts of E. coli K-12 [Waygood74, Waygood76], and recombinant enzyne has been overproduced and characterized [Valentini00]. A detailed kinetic analysis of pyruvate kinase I demonstrated the dependence of the reaction rate on the concentrations of various substrates, and the resulting sigmoid or hyperbolic curves. Several conformational states of the enzyme were defined [Markus80, Boiteux83].
The crystal structure of E. coli pyruvate kinase I has been determined at 2.50 Å resolution. Crystal structures of mutants R292D and R271L have also been determined at 1.80 Å and 2.80 Å resolution, respectively. The data suggested that during the quaternary structure transition from the inactive T-state to the active R-state the 12 domains of the tetramer change orientation. The domain interfaces couple changes in tertiary and quaternary structure to alterations in the fructose 1,6-bisphosphate and substrate binding sites [Mattevi95, Valentini00].
The oligomeric state of pyruvate kinase I in the presence of different allosteric effectors was investigated using steady-state kinetics. Analytical ultracentrifugation with fluorescence monitoring allowed detection of the enzyme at low nanomolar concentrations. The results showed that the dissociation constant is very low and that neither the substrates nor the allosteric effector affected the tetrameric state [Zhu10].
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].
Genes pykF and pykA are often used in metabolic engineering due to their importance in the control of metabolic flux in glycolysis. In various strains of E. coli the effects of single and double mutants of pykF and pykA on gene expression levels, enzyme activities, metabolite concentrations, glycolytic flux, and the production of useful compounds have been studied [Ponce, Gosset96, Ponce99, Zhu, Emmerling02, Siddiquee04, Al04, Ponce05, Sabido14, Zhang11b, Meza12, Peskov12, Soellner13, Rodriguez13, Weiner14]. Single and double knockouts of genes pykF and pykA can also affect plasmid DNA synthesis and cyclic AMP levels [Goncalves13, Wunderlich14].
Regulation of pykF by FruR in response to carbon source has been described [Bledig96]. A mutant in the carbon storage regulator gene csrA is deficient in pyruvate kinase I activity as well as some other glycolytic enzymes, but is not deficient in pyruvate kinase II activity [Sabnis95].
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 [Kri14].
PykF: "pyruvate kinase, fructose 1,6-diphosphate-activated" [Kornberg73]
Locations: cytosol, membrane
|Map Position: [1,753,722 -> 1,755,134] (37.8 centisomes, 136°)||Length: 1413 bp / 470 aa|
Molecular Weight of Polypeptide: 50.729 kD (from nucleotide sequence), 60.0 kD (experimental) [Waygood74 ]
Molecular Weight of Multimer: 240.0 kD (experimental) [Waygood74]
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)
|Biological Process:||GO:0006096 - glycolytic process
[UniProtGOA12, UniProtGOA11, GOA01, Ponce95]
GO:0009408 - response to heat [Kri14]
GO:0051289 - protein homotetramerization [Valentini79]
GO:0008152 - metabolic process [UniProtGOA11]
GO:0016310 - phosphorylation [UniProtGOA11]
|Molecular Function:||GO:0004743 - pyruvate kinase activity
[GOA01a, GOA01, Valentini00, Waygood74]
GO:0042802 - identical protein binding [Valentini79]
GO:0000166 - nucleotide binding [UniProtGOA11]
GO:0000287 - magnesium ion binding [GOA01]
GO:0003824 - catalytic activity [UniProtGOA11, GOA01]
GO:0005524 - ATP binding [UniProtGOA11]
GO:0016301 - kinase activity [UniProtGOA11]
GO:0016740 - transferase activity [UniProtGOA11]
GO:0030955 - potassium ion binding [GOA01]
GO:0046872 - metal ion binding [UniProtGOA11]
|Cellular Component:||GO:0005829 - cytosol
[DiazMejia09, Ishihama08, LopezCampistrou05, Lasserre06]
GO:0016020 - membrane [Lasserre06]
|MultiFun Terms:||metabolism → energy metabolism, carbon → fermentation|
|metabolism → energy metabolism, carbon → glycolysis|
|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: 188.8.131.52
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 glycolysis and Entner-Doudoroff , superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass , mixed acid fermentation , glycolysis II (from fructose 6-phosphate) , glycolysis I (from glucose 6-phosphate)
The enzyme requires both divalent (Mg2+, Mn2+) and monovalent (K+) cations. The 5'-diphosphates of guanosine, inosine, uridine and cytidine can also serve as phospho acceptors [Muirhead90, Waygood74].
Primary Physiological Regulators of Enzyme Activity: fructose 1,6-bisphosphate
|Sequence-Conflict||451 -> 470|
10/20/97 Gene b1676 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10804; confirmed by SwissProt match.
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
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
Emmerling02: Emmerling M, Dauner M, Ponti A, Fiaux J, Hochuli M, Szyperski T, Wuthrich K, Bailey JE, Sauer U (2002). "Metabolic flux responses to pyruvate kinase knockout in Escherichia coli." J Bacteriol 184(1);152-64. PMID: 11741855
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
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
Goncalves13: Goncalves GA, Prazeres DM, Monteiro GA, Prather KL (2013). "De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production." Appl Microbiol Biotechnol 97(2);611-20. PMID: 22885693
Gosset96: Gosset G, Yong-Xiao J, Berry A (1996). "A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis in Escherichia coli." J Ind Microbiol 17(1);47-52. PMID: 8987689
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
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
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
Mattevi95: Mattevi A, Valentini G, Rizzi M, Speranza ML, Bolognesi M, Coda A (1995). "Crystal structure of Escherichia coli pyruvate kinase type I: molecular basis of the allosteric transition." Structure 3(7);729-41. PMID: 8591049
Meza12: Meza E, Becker J, Bolivar F, Gosset G, Wittmann C (2012). "Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli." Microb Cell Fact 11;127. PMID: 22973998
Ponce: Ponce E, Martinez A, Bolivar F, Valle F (1998). "Stimulation of glucose catabolism through the pentose pathway by the absence of the two pyruvate kinase isoenzymes in Escherichia coli." Biotechnol Bioeng 58(2-3);292-5. PMID: 10191403
Ponce05: Ponce E, Garcia M, Munoz ME (2005). "Participation of the Entner-Doudoroff pathway in Escherichia coli strains with an inactive phosphotransferase system (PTS- Glc+) in gluconate and glucose batch cultures." Can J Microbiol 51(11);975-82. PMID: 16333337
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
Rodriguez13: Rodriguez A, Martinez JA, Baez-Viveros JL, Flores N, Hernandez-Chavez G, Ramirez OT, Gosset G, Bolivar F (2013). "Constitutive expression of selected genes from the pentose phosphate and aromatic pathways increases the shikimic acid yield in high-glucose batch cultures of an Escherichia coli strain lacking PTS and pykF." Microb Cell Fact 12;86. PMID: 24079972
Sabido14: Sabido A, Sigala JC, Hernandez-Chavez G, Flores N, Gosset G, Bolivar F (2014). "Physiological and transcriptional characterization of Escherichia coli strains lacking interconversion of phosphoenolpyruvate and pyruvate when glucose and acetate are coutilized." Biotechnol Bioeng 111(6);1150-60. PMID: 24375081
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
Soellner13: Soellner S, Rahnert M, Siemann-Herzberg M, Takors R, Altenbuchner J (2013). "Evolution of pyruvate kinase-deficient Escherichia coli mutants enables glycerol-based cell growth and succinate production." J Appl Microbiol 115(6);1368-78. PMID: 23957584
Somani77: Somani BL, Valentini G, Malcovati M (1977). "Purification and molecular properties of the AMP-activated pyruvate kinase from Escherichia coli." Biochim Biophys Acta 482(1);52-63. PMID: 193572
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
Valentini79: Valentini G, Iadarola P, Somani BL, Malcovati M (1979). "Two forms of pyruvate kinase in Escherichia coli. A comparison of chemical and molecular properties." Biochim Biophys Acta 570(2);248-58. PMID: 387087
Valentini93: Valentini G, Stoppini M, Iadarola P, Malcovati M, Ferri G, Speranza ML (1993). "Divergent binding sites in pyruvate kinases I and II from Escherichia coli." Biol Chem Hoppe Seyler 374(1);69-74. PMID: 8439398
Waygood74: Waygood EB, Sanwal BD (1974). "The control of pyruvate kinases of Escherichia coli. I. Physicochemical and regulatory properties of the enzyme activated by fructose 1,6-diphosphate." J Biol Chem 249(1);265-74. PMID: 4588693
Waygood75: Waygood EB, Rayman MK, Sanwal BD (1975). "The control of pyruvate kinases of Escherichia coli. II. Effectors and regulatory properties of the enzyme activated by ribose 5-phosphate." Can J Biochem 53(4);444-54. PMID: 236081
Waygood76: Waygood EB, Mort JS, Sanwal BD (1976). "The control of pyruvate kinase of Escherichia coli. Binding of substrate and allosteric effectors to the enzyme activated by fructose 1,6-bisphosphate." Biochemistry 15(2);277-82. PMID: 764863
Weiner14: Weiner M, Trondle J, Albermann C, Sprenger GA, Weuster-Botz D (2014). "Carbon storage in recombinant Escherichia coli during growth on glycerol and lactic acid." Biotechnol Bioeng 111(12);2508-19. PMID: 24902947
Wunderlich14: Wunderlich M, Taymaz-Nikerel H, Gosset G, Ramirez OT, Lara AR (2014). "Effect of growth rate on plasmid DNA production and metabolic performance of engineered Escherichia coli strains." J Biosci Bioeng 117(3);336-42. PMID: 24012107
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
Zhu10: Zhu T, Bailey MF, Angley LM, Cooper TF, Dobson RC (2010). "The quaternary structure of pyruvate kinase type 1 from Escherichia coli at low nanomolar concentrations." Biochimie 92(1);116-20. PMID: 19800933
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
©2015 SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025-3493