View source for Pyruvate, water dikinase

{{infobox enzyme
| Name       = pyruvate, water dikinase
| EC_number  = 2.7.9.2
| CAS_number = 9013-09-6
| GO_code    = 0008986
| image      =
| width      = 
| caption    =  
}}
In [[enzymology]], a '''pyruvate, water dikinase''' ([[Enzyme Commission number|EC]] 2.7.9.2) is an [[enzyme]] that [[Catalysis|catalyzes]] the [[chemical reaction]]

: ATP + pyruvate + H<sub>2</sub>O  <math>\rightleftharpoons</math> AMP + phosphoenolpyruvate + phosphate

The 3 [[Substrate (biochemistry)|substrates]] of this enzyme are [[Adenosine triphosphate|ATP]], [[pyruvate]], and [[Water|H<sub>2</sub>O]], whereas its 3 [[Product (chemistry)|products]] are [[Adenosine monophosphate|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.<ref name=":05">{{Cite journal |last1=Hutchins |first1=A. M. |last2=Holden |first2=J. F. |last3=Adams |first3=M. W. |date=2001 |title=Phosphoenolpyruvate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus |journal=Journal of Bacteriology |volume=183 |issue=2 |pages=709–715 |doi=10.1128/JB.183.2.709-715.2001 |issn=0021-9193 |pmid=11133966|pmc=94928 }}</ref><ref name=":14">{{Cite journal |last1=Sakuraba |first1=H. |last2=Utsumi |first2=E. |last3=Kujo |first3=C. |last4=Ohshima |first4=T. |date=1999-04-01 |title=An AMP-dependent (ATP-forming) kinase in the hyperthermophilic archaeon Pyrococcus furiosus: characterization and novel physiological role |url=https://pubmed.ncbi.nlm.nih.gov/10087174/ |journal=Archives of Biochemistry and Biophysics |volume=364 |issue=1 |pages=125–128 |doi=10.1006/abbi.1999.1121 |issn=0003-9861 |pmid=10087174}}</ref>

This enzyme belongs to the family of [[transferase]]s, to be specific, those transferring phosphorus-containing groups ([[phosphotransferase]]s) with paired acceptors ([[dikinase]]s). The [[List of enzymes|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 (biochemistry)|cofactor]], [[manganese]].

== 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 [[Thermophile|thermophilic]] or [[Hyperthermophile|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.{{Citation needed|date=October 2023}}

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 [[heterotroph]]ic 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.{{Citation needed|date=October 2023}}

== Structure ==
As of 2023, only one [[Tertiary structure|structure]] has been solved for this class of enzymes, with the [[Protein Data Bank|PDB]] accession code [https://www.rcsb.org/structure/2OLS 2OLS]. The crystalline structure from ''[[Neisseria meningitidis]]'' was computed through [[X-ray crystallography|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.<ref>{{Cite web |last1=Zhang |first1=R |last2=Duggan |first2=E |last3=Bargassa |first3=M |last4=Joachimiak |first4=A |date=2007 |title=RCSB PDB - 2OLS: The crystal structure of the phosphoenolpyruvate synthase from Neisseria meningitidis |url=https://www.rcsb.org/structure/2OLS |website=www.rcsb.org}}</ref>

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.<ref name=":05" /> 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.<ref name=":05" /> 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.<ref name=":05" />

== 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.<ref name=":14" /><ref name=":23">{{Cite journal |last1=Sakuraba |first1=H. |last2=Utsumi |first2=E. |last3=Schreier |first3=H. J. |last4=Ohshima |first4=T. |date=2001 |title=Transcriptional regulation of phosphoenolpyruvate synthase by maltose in the hyperthermophilic archaeon, Pyrococcus furiosus |url=https://pubmed.ncbi.nlm.nih.gov/16233068/ |journal=Journal of Bioscience and Bioengineering |volume=92 |issue=2 |pages=108–113 |doi=10.1263/jbb.92.108 |issn=1389-1723 |pmid=16233068}}</ref> 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]].<ref name=":33">{{Cite journal |last1=Schäfer |first1=T. |last2=Schönheit |first2=P. |date=1993 |title=Gluconeogenesis from pyruvate in the hyperthermophilic archaeon Pyrococcus furiosus: involvement of reactions of the Embden-Meyerhof pathway. |url=https://link.springer.com/article/10.1007/BF00290918 |journal=Archives of Microbiology |volume=159 |issue=4 |pages=354–363 |doi=10.1007/BF00290918 |bibcode=1993ArMic.159..354S |via=Springer Link}}</ref> For their metabolism, ''Pyrococcus furiosus'' uses carbon sources like [[maltose]], [[cellobiose]], [[laminarin]], and [[starch]]es in this sugar metabolic pathway to produce energy for the organism.<ref name=":23" />

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.<ref name=":05" /> 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.<ref name=":05" /> 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.<ref name=":05" />

== 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.<ref name=":23" /> 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.<ref name=":14" /><ref name=":23" /> Pyruvate, water dikinase in ''Pyrococcus furiosus'' is sensitive to oxygen, with no enzyme activity measured in aerobic conditions.<ref name=":33" /> The purified pyruvate, water dikinase in ''Pyrococcus furiosus'' has a pH optimum between 6.5 and 9, and a temperature optimum around 90˚C.<ref name=":05" /><ref name=":14" /> In the PEP formation reaction, pyruvate has an apparent  K<sub>m</sub> of 0.11mM, apparent k<sub>cat</sub> of 1,573(s<sup>−1</sup>) and apparent k<sub>cat</sub>/K<sub>m</sub> of 1.43 x 10^4 (mM<sup>−1</sup>• s-<sup>1</sup>), and ATP has an apparent Km of 0.39mM, apparent kcat of 1,326(s<sup>−1</sup>) and apparent kcat/Km of 3.40 x 10^3 (mM<sup>−1</sup> • s<sup>−1</sup>). In the pyruvate formation reaction, PEP has an apparent  K<sub>m</sub> of 0.40mM, apparent k<sub>cat</sub> of 12.6(s<sup>−1</sup>) and apparent k<sub>cat</sub>/K<sub>m</sub> of 31.5 (mM<sup>−1</sup> • s<sup>−1</sup>), AMP has an apparent  K<sub>m</sub> of 1.00mM, apparent k<sub>cat</sub> of 8.7(s<sup>−1</sup>) and apparent k<sub>cat</sub>/K<sub>m</sub> of 8.7 (mM<sup>−1</sup> • s<sup>−1</sup>), and phosphate has an apparent  K<sub>m</sub> of 38.4mM, apparent k<sub>cat</sub> of 11.9(s<sup>−1</sup>) and apparent k<sub>cat</sub>/K<sub>m</sub> of 0.315(mM<sup>−1</sup> • s<sup>−1</sup>).<ref name=":05" /> The equilibrium constant K<sub>eq</sub> 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.<ref name=":14" />

==References==
{{reflist|1}}
* {{cite journal | vauthors = Berman KM, Cohn M | date = 1970 | title = Phosphoenolpyruvate synthetase of Escherichia coli. Purification, some properties, and the role of divalent metal ions | journal = J. Biol. Chem.  | volume = 245 | pages = 5309&ndash;18  | pmid = 4319237 | issue = 20 | doi = 10.1016/S0021-9258(18)62756-5 | doi-access = free }}
* {{cite journal | vauthors = Berman KM, Cohn M | date = 1970 | title = Phosphoenolpyruvate synthetase. Partial reactions studied with adenosine triphosphate analogues and the inorganic phosphate-H2 18O exchange reaction | journal = J. Biol. Chem.  | volume = 245 | pages = 5319&ndash;25  | pmid = 4319238 | issue = 20 | doi = 10.1016/S0021-9258(18)62757-7 | doi-access = free }}
* {{cite journal | vauthors = Cooper RA, Kornberg HL | date = 1965 | title = Net formation of phosphoenolpyruvate from pyruvate by Escherichia coli | journal = Biochim. Biophys. Acta  | volume = 104 | pages = 618&ndash;20  | pmid = 5322808 | issue = 2 | doi=10.1016/0304-4165(65)90374-0}}
* {{cite book |author1 = Cooper RA |author2 =Kornberg HL |title =Citric Acid Cycle |chapter =Phosphoenolpyruvate synthetase | date = 1969 | volume = 13 | pages = 309&ndash;314 | doi = 10.1016/0076-6879(69)13053-0 | series = Methods in Enzymology | isbn = 978-0-12-181870-8 }}

{{Kinases}}
{{Enzymes}}
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[[Category:EC 2.7.9]]
[[Category:Manganese enzymes]]
[[Category:Enzymes of known structure]]