Pyruvate, water dikinase
In enzymology, a pyruvate, water dikinase (EC 2.7.9.2) is an enzyme that catalyzes the chemical reaction
- ATP + pyruvate + H2O AMP + phosphoenolpyruvate + phosphate
| pyruvate, water dikinase | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Identifiers | |||||||||
| EC no. | 2.7.9.2 | ||||||||
| CAS no. | 9013-09-6 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDB PDBe PDBsum | ||||||||
| Gene Ontology | AmiGO / QuickGO | ||||||||
| |||||||||
The 3 substrates of this enzyme are ATP, pyruvate, and H2O, whereas its 3 products are AMP, phosphoenolpyruvate, and phosphate. This reaction catalyzed by pyruvate, water dikinase can run in both directions, but has a strong preference for AMP, phosphate, and phosphoenolpyruvate as substrate and typically runs in the ATP producing direction [1][2].
This enzyme belongs to the family of transferases, to be specific, those transferring phosphorus-containing groups (phosphotransferases) with paired acceptors (dikinases). The systematic name of this enzyme class is ATP:pyruvate, water phosphotransferase. Other names in common use include phosphoenolpyruvate synthase, pyruvate-water dikinase (phosphorylating), PEP synthetase, PEP synthase, PEPS, phoephoenolpyruvate synthetase, phosphoenolpyruvic synthase, and phosphopyruvate synthetase. This enzyme participates in pyruvate metabolism and reductive carboxylate cycle (CO2 fixation). It employs one cofactor, manganese.
EC Number
Enzymes are grouped and classified by their Enzyme Commission number, which is based on what reaction the enzyme catalyzes. The EC number for pyruvate, water dikinase is EC 2.7.9.2. Each number corresponds to a different level of enzyme reaction classification: EC 2 enzymes are Transferases, EC 2.7 enzymes are transferases that transfer phosphorus-containing groups, EC 2.7.9 enzymes are phosphotransferases with paired acceptors, and EC 2.7.9.2 is the unique EC number code for the enzyme reaction that pyruvate, water dikinase catalyzes.
Studied Organisms
According to the BRENDA database, pyruvate, water dikinase has been studied in nine unique bacterial and archaea species under a wide range of names. Many of the studied organisms are thermophilic or hyperthermophilic, meaning they live and function in very high temperatures in their natural environments, and have been found in hot springs, volcanos, and deep sea hydrothermal vents.
One of the most widely studied organisms for pyruvate, water dikninase is Pyrococcus furiosus. Pyrococcus furiosus is a deep sea hyperthermophilic archaea that is commonly found living in extremely hot waters around hydrothermal vents. This species is heterotrophic and anaerobic (grows and metabolizes without the presence of oxygen), and has an optimal growth temperature of 100˚C. The enzymes and proteins in this species are studied and of note because of their thermal stability. Pyrococcus furiosus organisms use the fermentation of carbohydrates and glycolysis to produce energy.
Structure
As of 2023, only one structure has been solved for this class of enzymes, with the PDB accession code 2OLS. The crystalline structure from Neisseria meningitidis was computed through x-ray diffraction techniques at a resolution of 2.40 Å. Pyruvate, water dikinase in Neisseria meningitidis is 794 amino acids in length and has two active sites: one at at position 422 and position 752[3].
In Pyrococcus furiosus, the pyruvate, water dikinase enzyme has a subunit molecular mass of 92 kDa, and each subunit contains one calcium and one phosphorus atom[1]. This enzyme has a octomeric structure, meaning that pyruvate, water dikinase in Pyrococcus furiosus is an oligomer protein consisting of eight subunits in its quaternary structure[1]. This eight subunit protein structure might help this enzyme function at high temperatures. This enzyme comes in two protein types, one phosphorylated and one non phosphorylated version. The N terminal amino acid sequences the same in both versions, which shows these two forms are phosphorylated and non phosphorylated versions of pyruvate, water dikinase[1].
Reaction Pathway and Biological Function
In Pyrococcus furiosus, pyruvate, water dikinase is the enzyme that catalyzes the first step of gluconeogenesis from pyruvate in the modified Embden-Meyerhof pathway (M-EMP) and is an important ATP producing reaction in the metabolism pathway[2][4]. The modified Embden-Meyerhof pathway is a glycolytic pathway that converts glucose into pyruvate and energy products for the cell. This enzyme participates in catalyzing reactions that are important for both gluconeogenesis and the reverse, glycolysis[5]. For their metabolism, Pyrococcus furiosus uses carbon sources like maltose, cellobiose, laminarin, and starches in this sugar metabolic pathway to produce energy for the organism[4].
Pyruvate, water dikinase in Pyrococcus furiosus primarily catalyzes the reaction that goes from phosphoenolpyruvate and AMP to pyruvate and ATP, but can also catalyze the reverse reaction[1]. This reaction is thought to be important because it converts AMP into usable ATP energy during this sugar M-EMP metabolism. Two sugar kinase enzymes (glucokinase and phosphofructokinase) were found in the M-EMP pathway in Pyrococcus furiosus that catalyze the reaction that used ADP and produces AMP. In order for the AMP to be usable as ATP in the cell, the pyruvate, water dikinase enzyme catalyzes the phosphate dependent formation of pyruvate reaction pathway to convert AMP to ATP. This enzyme uses phosphoenolpyruvate as the phosphoryl group donor and then forms ATP in the presence of phosphate.
One study determined that pyruvate, water dikinase in Pyrococcus furiosus can act in a futile cycle between phosphoenolpyruvate and pyruvate as substrates/products[1]. These two reactions can run through the metabolic pathways at the same time in opposite directions, which will dissipate energy as heat without other effects. This can remove unwanted energy, as the energy produced from glycolysis is much more than the energy required for growth and cellular repairs. This is possibly a mode of "energy spilling" in Pyrococcus furiosus. This is in part hypothesized because of to the high concentrations of this enzyme (~5% of protein in the cytoplasm) in Pyrococcus furiosus cells[1].
Enzyme Kinematics
The hyperthermostable pyruvate, water dikinase enzyme in Pyrococcus furiosus is encoded by the mlrA gene, which was found to be regulated by at least in part by maltose at a transcription level[4]. Pyruvate, water dikinase catalyzes the reaction that converts phosphoenolpyruvate, AMP, and phosphate to pyruvate, ATP, and water. This enzyme also catalyzes the reverse reaction, but reaction rates and equilibrium constants show that the ATP production reaction direction is highly favorable[2][4]. Pyruvate, water dikinase in Pyrococcus furiosus is sensitive to oxygen, with no enzyme activity measured in aerobic conditions[5]. The purified pyruvate, water dikinase in Pyrococcus furiosus has a pH optimum between 6.5 and 9, and a temperature optimum around 90˚C[1][2]. In the PEP formation reaction, pyruvate has an apparent Km of 0.11mM, apparent kcat of 1,573(s-1) and apparent kcat/Km of 1.43 x 10^4 (mM-1• s-1), and ATP has an apparent Km of 0.39mM, apparent kcat of 1,326(s-1) and apparent kcat/Km of 3.40 x 10^3 (mM-1 • s-1). In the pyruvate formation reaction, PEP has an apparent Km of 0.40mM, apparent kcat of 12.6(s-1) and apparent kcat/Km of 31.5 (mM-1 • s-1), AMP has an apparent Km of 1.00mM, apparent kcat of 8.7(s-1) and apparent kcat/Km of 8.7 (mM-1 • s-1), and phosphate has an apparent Km of 38.4mM, apparent kcat of 11.9(s-1) and apparent kcat/Km of 0.315(mM-1 • s-1)[1]. The equilibrium constant Keq of the reaction is 1.07 at 50˚C, and the change in Gibbs free energy (ΔG˚) is -0.04 kcal/mol at experimental conditions[2].
References
- Hutchins, A. M.; Holden, J. F.; Adams, M. W. (2001). "Phosphoenolpyruvate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus". Journal of Bacteriology. 183 (2): 709–715. doi:10.1128/JB.183.2.709-715.2001. ISSN 0021-9193. PMID 11133966.
- Sakuraba, H.; Utsumi, E.; Kujo, C.; Ohshima, T. (1999-04-01). "An AMP-dependent (ATP-forming) kinase in the hyperthermophilic archaeon Pyrococcus furiosus: characterization and novel physiological role". Archives of Biochemistry and Biophysics. 364 (1): 125–128. doi:10.1006/abbi.1999.1121. ISSN 0003-9861. PMID 10087174.
- Zhang, R; Duggan, E; Bargassa, M; Joachimiak, A (2007). "RCSB PDB - 2OLS: The crystal structure of the phosphoenolpyruvate synthase from Neisseria meningitidis". www.rcsb.org.
- Sakuraba, H.; Utsumi, E.; Schreier, H. J.; Ohshima, T. (2001). "Transcriptional regulation of phosphoenolpyruvate synthase by maltose in the hyperthermophilic archaeon, Pyrococcus furiosus". Journal of Bioscience and Bioengineering. 92 (2): 108–113. doi:10.1263/jbb.92.108. ISSN 1389-1723. PMID 16233068.
- Schäfer, T.; Schönheit, P. (1993). "Gluconeogenesis from pyruvate in the hyperthermophilic archaeon Pyrococcus furiosus: involvement of reactions of the Embden-Meyerhof pathway". Archives of Microbiology. 159: 354–363 – via Springer Link.
- Berman KM, Cohn M (1970). "Phosphoenolpyruvate synthetase of Escherichia coli. Purification, some properties, and the role of divalent metal ions". J. Biol. Chem. 245 (20): 5309–18. PMID 4319237.
- Berman KM, Cohn M (1970). "Phosphoenolpyruvate synthetase. Partial reactions studied with adenosine triphosphate analogues and the inorganic phosphate-H2 18O exchange reaction". J. Biol. Chem. 245 (20): 5319–25. PMID 4319238.
- Cooper RA, Kornberg HL (1965). "Net formation of phosphoenolpyruvate from pyruvate by Escherichia coli". Biochim. Biophys. Acta. 104 (2): 618–20. doi:10.1016/0304-4165(65)90374-0. PMID 5322808.
- Cooper RA; Kornberg HL (1969). "Phosphoenolpyruvate synthetase". Methods Enzymol. Methods in Enzymology. 13: 309–314. doi:10.1016/0076-6879(69)13053-0. ISBN 978-0-12-181870-8.